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| J Biol Chem, 1991 Jul 15, 266(20), 12964 - 70 Cloning and expression of a yeast protein tyrosine phosphatase; Guan KL et al.; To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR) . Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment . The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases . The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein . This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein . The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1 . This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step . The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate . Gene disruption of yeast PTP1 has no visible effect on vegetative growth. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6264 - 8 Isoprenoid modification of rab proteins terminating in CC or CXC motifs; Khosravi-Far R et al.; Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization . rab1b and rab3a proteins expressed in insect cells incorporated a product of {3H}mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups . Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6) . Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups . A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro . However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b . Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid) . Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6072 - 6 Trans-acting transposase mutant from Tn5; DeLong A et al.; Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh . The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study . A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation . A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized . The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp . This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis . Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans . These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 5979 - 83 Proteinase trapping: screening for viral proteinase mutants by alpha complementation; Liebig HD et al.; Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus . This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18 . When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment . However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment . After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes . Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector . alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein . This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity. Cell, 1991 Jul 12, 66(1), 121 - 8 Three-dimensional structure of yeast RNA polymerase II at 16 A resolution; Darst SA et al.; The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution . The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E . coli RNA polymerase . The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide . The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively . A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit . These results provide a structural basis for analyses of the transcription process and its regulation. J Chromatogr, 1991 Jul 12, 548(1-2), 165 - 78 Characterization of synthetic macroporous ion-exchange resins in low-pressure cartridges and columns . Evaluation of the performance of Macro-Prep 50 S resin in the purification of anti-Klenow antibodies from goat serum; Dunn L et al.; Three ion-exchange materials and one hydrophobic-interaction chromatography packing, based on a rigid macroporous polymer with large, relatively uniform pores, have been evaluated for low-pressure liquid chromatography of antibodies . These sorbents have high capacities for both small and large proteins and are mechanically, chemically, and thermally stable . Macro-Prep 50 S . CM and Q ion-exchange materials are strongly acidic, weakly acidic, and strongly basic, respectively . Protein binding and recovery, pressure-flow properties, and chemical and thermal stability were determined for each sorbent . A rapid, two-step method for the purification of anti-Klenow antibodies from goat serum was developed, based on the Macro-Prep 50 S strong-acid cation-exchange material and the Econo-Pac HIC prepacked hydrophobic-interaction cartridge. Science, 1991 Jul 12, 253(5016), 164 - 70 A method to identify protein sequences that fold into a known three-dimensional structure; Bowie JU et al.; The inverse protein folding problem, the problem of finding which amino acid sequences fold into a known three-dimensional (3D) structure, can be effectively attacked by finding sequences that are most compatible with the environments of the residues in the 3D structure . The environments are described by: (i) the area of the residue buried in the protein and inaccessible to solvent; (ii) the fraction of side-chain area that is covered by polar atoms (O and N); and (iii) the local secondary structure . Examples of this 3D profile method are presented for four families of proteins: the globins, cyclic AMP (adenosine 3',5'-monophosphate) receptor-like proteins, the periplasmic binding proteins, and the actins . This method is able to detect the structural similarity of the actins and 70- kilodalton heat shock proteins, even though these protein families share no detectable sequence similarity. Mol Cell Endocrinol, 1991 Jul, 78(3), 163 - 70 A synthetic peptide corresponding to hFSH-beta-(81-95) has thioredoxin-like activity; Grasso P et al.; The thioredoxin-like activity of human follicle stimulating hormone (hFSH), hFSH-beta-(83-88) peptide amide (hFSH-beta-(83-88) which has a sequence similar to the thioredoxin active center (-His-Cys-Gly-Lys-Cys-Asp-)) and thioredoxin-(31-36)-peptide amide (TD-(31-36) which contains the redox-active dithiol of thioredoxin (-Trp-Cys-Gly-Pro-Cys-Lys-)) was characterized by their ability to reactivate reduced and denatured bovine pancreatic ribonuclease (RNase) . This assay reflects the recently recognized ability of thioredoxin to catalyze disulfide bond formation in proteins . Compared to uncatalyzed refolding of reduced, denatured substrate, hFSH was approximately 10-fold more active than thioredoxin on a molar basis . The catalytic activity of hFSH-beta-(83-88) and TD-(31-36) was equivalent to that of an equimolar concentration of thioredoxin . Screening of 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit indicated that hFSH-beta-(81-95), which contains the sequence similar to the thioredoxin active center within a receptor-binding region of the hFSH-beta-subunit, possesses strong thioredoxin-like activity and was more active than an equimolar concentration of thioredoxin . In contrast, hFSH-beta-(33-53), a thiol-containing peptide which corresponds to a second FSH receptor-binding domain but lacks the sequence similar to the thioredoxin active center, was inactive . Synthetic peptide amides corresponding to other regions of hFSH-beta-subunit were less effective than hFSH-beta-(81-95) in reactivating reduced and denatured RNase . Our data provide evidence that the recently reported thioredoxin-like catalytic activity of FSH may be due, at least in part, to the redox-active dithiol present within a receptor-binding domain of its beta-subunit, and thus may have a physiological role in receptor binding or signal transduction. Nature, 1991 Jul 11, 352(6331), 172 - 4 Convergent evolution of similar function in two structurally divergent enzymes; Kuriyan J et al.; An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase . Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin . Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site . Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other . We have determined the crystal structure of E . coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure . The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system . This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently. Nucleic Acids Res, 1991 Jul 11, 19(13), 3533 - 41 Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent; Prats AC et al.; The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions . In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions . In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination . These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity . However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10 . These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly . Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions. Nucleic Acids Res, 1991 Jul 11, 19(13), 3499 - 506 Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence; Matteson RJ et al.; In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet . Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA . The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes . The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency . On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons . Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs. Nucleic Acids Res, 1991 Jul 11, 19(13), 3577 - 81 Characterization of a protein binding sequence in the promoter region of the 16S rRNA gene of the spinach chloroplast genome; Baeza L et al.; By means of mobility-shift assays and Exonuclease III mapping we have determined a 14 bp sequence (named CDF2 binding site) located in front of the 16S rRNA initiation start site which is protected by a spinach chloroplast extract . This region does not include neither one of the two '-35' nor of the two '-10' E . coli-like promoter elements which are recognised by E . coli RNA polymerase in vitro . The CDF2 binding site is specifically recognized by two small polypeptides which migrate corresponding to 35 and 33 kDa respectively as shown by UV cross-linking experiments . In vivo transcription initiation of the 16S rRNA gene occurs 13 nucleotides downstream of the 14 bp sequence and is different from the transcription start site which is used by E.coli polymerase in vitro. Nucleic Acids Res, 1991 Jul 11, 19(13), 3511 - 6 Amino acid misincorporation during high-level expression of mouse epidermal growth factor in Escherichia coli; Scorer CA et al.; To determine whether the high-level expression of foreign proteins in Escherichia coli can lead to frequent translational errors, we analyzed amino acid misincorporation in mouse epidermal growth factor (mEGF) produced as a TrpE fusion protein . The mEGF DNA does not encode phenylalanine and determining the phenylalanine content of the purified protein will measure missense errors . Using this approach, we found an error frequency of about 1 in 40 for codons differing by a single base from those for phenylalanine . This is at least ten times higher than the error rate found for normal E . coli protein synthesis and may be due to limiting supply of charged tRNAs and GTP, brought about by the high-level production of the heterologous protein . The unexpectedly high error rate has implications for the clinical use of E . coli-derived therapeutic proteins. Nucleic Acids Res, 1991 Jul 11, 19(13), 3653 - 60 Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe; Dubreuil YL et al.; The structure in solution of an RNA fragment (218 nucleotides long) containing part of E . coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U) . In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules . An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content . Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U . The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size . The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing . 17 of the 41 possible s4U positions lead to detectable bridges . These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed . The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected. Nucleic Acids Res, 1991 Jul 11, 19(13), 3517 - 24 The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine; Doring T et al.; Intact 50S ribosomal subunits from E.coli were cross-linked with the symmetrical bifunctional reagent bis-(2-chloroethyl)-methylamine . After deproteinization, selected regions of the 23S RNA were excised by treatment with ribonuclease H in the presence of appropriate complementary decadeoxynucleotides, and screened for the presence of intra-RNA cross-links by two-dimensional gel electrophoresis . Individual isolated cross-linked RNA fragments were analysed by our established procedures . Sixteen intra-RNA cross-links were identified, three of which corresponded to those previously published . The thirteen 'new' cross-links were localized in the 23S RNA at positions 774-78 linked to 792-94, 876-79 linked to 899-900, 979-81 or 983-84 to 2029, 1715 to 1743-46, 1911-21 to 1964, 1933 to 1966, 2032 to 2054-55, 2112 to 2169-71, 2116-17 to 2163-67, 2128-32 to 2156-59, 2392-93 to 2422-23, 2737-38 to 2763-66, and 2791 to 2890 . These results are discussed in the context of three-dimensional model-building studies with the 23S RNA, with particular reference to the environment of the 'active centre' of the 50S subunit. Nucleic Acids Res, 1991 Jul 11, 19(13), 3629 - 32 MutM, a protein that prevents G.C----T.A transversions, is formamidopyrimidine-DNA glycosylase; Michaels ML et al.; We have cloned chromosomal DNA bordering an insert that inactivates mutM . Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) . An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis . Thus, we conclude that MutM is actually Fapy-DNA glycosylase . mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions . This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG). Nucleic Acids Res, 1991 Jul 11, 19(13), 3613 - 8 DNA helicase IV from HeLa cells; Tuteja N et al.; Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized . The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA . From 440g of HeLa cells we obtained 0.31 mg of pure protein . Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities . The apparent molecular weight is 100 kDa . It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP . The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly {G} (as phosphate) . The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res . 18, 6785-6792, 1990) . It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure . Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid. Biochim Biophys Acta, 1991 Jul 10, 1093(2-3), 135 - 43 The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway; Nishikawa S et al.; SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus . To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein . This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate . We examined the subcellular localization of Sar1p using this antibody . In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER . The soluble form of Sar1p was also detected when the protein was overproduced . Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant . Membrane association of Sar1p was shown to be very light . Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely . From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic. Biochim Biophys Acta, 1991 Jul 10, 1093(2-3), 216 - 22 Hepatic phosphatidylinositol kinase activity in continuously endotoxemic rats; Rodriguez de Turco EB et al.; The activity of phosphatidylinositol (PI) kinase and the content and fatty acid composition of inositol phospholipids (IPLs) were analyzed in the livers of rats that had been continuously infused with Escherichia coli endotoxin (ET) or saline for 30 h . Maximal enzymatic activity in total liver membrane fractions was observed in the presence of 1 mM ATP, 20 mM MgCl2, exogenously added 0.3 mM PI and Triton X-100 (0.25%) . The activity of PI kinase for endogenous and exogenous PI was 43 and 79% higher respectively, in ET- as compared with saline-infused rats . The Km of the enzyme for ATP was not altered (0.175 mM), while the apparent Vmax was higher for ET- as compared with saline-infused rats (0.48 and 0.38 nmol of phosphatidylinositol 4-phosphate formed/mg protein per min, respectively) . The ET-induced higher activity of PI kinase was paralleled by a 68-78% increase in the content of polyphosphoinositides (PPI), while PI content was unchanged . All IPLs from livers of endotoxemic rats had a lower content of arachidonic acid . We demonstrate for the first time that ET can directly and/or indirectly stimulate the net synthesis of PPI in liver cells . This effect could serve to modulate the PPI derived signals by increasing the availability of the substrate phosphatidylinositol 4,5-bisphosphate. Biochemistry, 1991 Jul 9, 30(27), 6775 - 9 Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence; Loewenthal R et al.; Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues . The spectrum is dominated by the contribution of Trp-35 . The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75 . This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989) . This histidine residue is close to Trp-94 . The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction . The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein . Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94. Biochemistry, 1991 Jul 9, 30(27), 6721 - 6 Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Lolkema JS et al.; Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased . The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG . The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence . Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII . When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme . The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII {Lolkema J . S., ten Hoeve-Duurkens, R . H., Swaving Dijkstra, D., & Robillard, G . T . (1991) Biochemistry (preceding paper in this issue)} . Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant . No effect on the binding properties was observed . In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr. Biochemistry, 1991 Jul 9, 30(27), 6716 - 21 Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Lolkema JS et al.; Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr . The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol . The latter was 2-3 orders of magnitude slower . The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50% . The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent . Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space . A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments . The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol. Biochemistry, 1991 Jul 9, 30(27), 6788 - 95 Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli; Yang XY et al.; The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon . The amino-terminal sequence of this protein had been established by Edman degradation . The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis . Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively . The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed {Yang, S.-Y., et al . (1990) J . Biol . Chem . 265, 10424-10429}, was corroborated . The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593 . A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product . Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl-CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl-CoA isomerase and suggest that the genes of the E . coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene. FEBS Lett, 1991 Jul 8, 285(1), 31 - 4 Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli; Yoshioka K et al.; Drosophila copia protease is likely to be encoded in the gag gene . We have expressed copia gag polyprotein precursor in E . coli . The gag precursor was correctly processed to generate a unique laminate structure in E . coli . The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor . Furthermore, the laminate structure was not found in E . coli expressing the mutant precursor . These results indicate that the protease is involved in cleaving the gag precursor itself . Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor. FEBS Lett, 1991 Jul 8, 285(1), 127 - 31 A chemically cross-linked nonlinear proOmpA molecule can be translocated into everted membrane vesicles of Escherichia coli in the presence of the proton motive force; Tani K et al.; The chemical cross-linking between the two cysteine residues at positions + 290 and + 302 of proOmpA was performed with N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine . In the absence of the proton motive force (delta muH+), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates . In the presence of delta mu H+, the cross-linked proOmpA was completely translocated . The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker . It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated. J Biol Chem, 1991 Jun 25, 266(18), 11610 - 3 The priB gene encoding the primosomal replication n protein of Escherichia coli; Allen GC Jr et al.; The gene encoding protein n of the Escherichia coli primosome has been discovered in the rpsF-rpsR-rplI ribosomal protein operon and designated priB . The low copy number of PriB protein and the distinctive codon usage of its gene argue against its being a ribosomal protein . A strain which overproduces PriB was constructed and has been used to purify the protein to homogeneity . The overproduced protein behaves like that purified from wild-type cells. J Mol Biol, 1991 Jul 5, 220(1), 19 - 33 Initiation of the UvrABC nuclease cleavage reaction . Efficiency of incision is not correlated with UvrA binding affinity; Snowden A et al.; The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex . We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites . To investigate these differences, quantitative DNase I footprinting titration studies were performed . The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions . It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively . These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage . It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction. J Mol Biol, 1991 Jul 5, 220(1), 13 - 6 Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli; Abeysinghe SI et al.; Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme . Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant . The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively . Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits. J Biol Chem, 1991 Jul 5, 266(19), 12759 - 65 Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis; Yang YF et al.; By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined . Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones . Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT) . Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26 . The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces . The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure . The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme. J Biol Chem, 1991 Jul 5, 266(19), 12469 - 73 Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1; Kelly GM et al.; Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis . There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1 . Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions . An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM . Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding . Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1 . Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development. J Biol Chem, 1991 Jul 5, 266(19), 12439 - 41 Spermidine acetylation in response to a variety of stresses in Escherichia coli; Carper SW et al.; Heat shock, cold shock, ethanol, and alkaline shift, but not hydrogen peroxide, stimulate the accumulation of monoacetylspermidine in Escherichia coli . Acetylation occurs with nearly equal frequencies at both the N1 and N8 positions of this ubiquitous polycation . Spermidine acetylation does not appear to be associated with known stress regulons, such as htpR, oxyR, and SOS . E . coli, capable of acetylating spermidine, constitutively express a spermidine acetyltransferase activity during all phases of growth, and this activity is unaffected by cold shock . A mutant strain, incapable of acetylating spermidine, does not express this enzyme activity but grows at an identical rate as the parent strain at 37 degrees C . These results demonstrate that the monoacetylation of spermidine in E . coli is regulated by some mechanism other than a stress-inducible acetyltransferase and is not essential for growth of these cells . They suggest that polyamine acetylation is involved in the responses of these organisms to a variety of chemical and physical stresses. J Biol Chem, 1991 Jul 5, 266(19), 12228 - 33 Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis; Dong Q et al.; Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1 . The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification . The other substrate, aspartate, had no such effect even at a high concentration . In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed . Stoichiometry studies with {7-14C}phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP . Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification . This arginine has been changed to leucine by site-directed mutagenesis . The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme . In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4) . On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis. J Biol Chem, 1991 Jul 5, 266(19), 12495 - 501 Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus . Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution; Grimm B et al.; Glutamate 1-semialdehyde aminotransferase (GSA-AT) is the last enzyme in the C5 pathway converting glutamate into the tetrapyrrole precursor delta-aminolevulinate in plants, algae, and several bacteria . Sequence analysis of the genes encoding GSA-AT in barley, Synechococcus, and Escherichia coli revealed 50-70% similarity in the primary structures of the proteins . The enzyme is inhibited rapidly by gabaculine when added in approximately stoichiometric amounts with the enzyme . A gabaculine-tolerant Synechococcus strain, GR6, was found to produce a GSA-AT less sensitive to the inhibitor . Accordingly, the mutant gene was isolated and sequenced . In comparison with the wild-type gene it contains a deletion of nine nucleotides (position 12-20) and a guanine to adenine substitution (position 743) . This resulted in the loss of the amino acids serine, proline, and phenylalanine (position 5-7) close to the NH2 terminus of the enzyme and an exchange of Met-248 for isoleucine in the middle of the polypeptide chain . Wild-type and mutant GSA-AT were expressed in E . coli and purified close to homogeneity . Although the specific activity of the mutant GSA-AT was only one-fifth of the wild type, it displayed a 100-fold increased resistance to gabaculine . Peaks in the absorption spectrum of the purified recombinant GSA-ATs at 335 and 417 nm are typical of a transaminase containing a B6 cofactor . Incubation with substrate and with inhibitor induced spectral changes characteristic of other gabaculine-sensitive, B6-requiring enzymes. J Biol Chem, 1991 Jul 5, 266(19), 12722 - 33 A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the kappa B enhancer element; Ostrowski J et al.; The immunoglobulin kappa light chain enhancer, kappa B, is an important cis-acting transcriptional element . kappa B binds a number of proteins including the members of the ubiquitous NF-kappa B family of transcription factors . Agarose beads coupled to a double-stranded oligonucleotide containing the kappa B motif were used to isolate a 65-kDa predominantly nuclear phosphoprotein . Southwestern blot analysis demonstrated that this phosphoprotein can bind the kappa B element directly and specifically . This kappa B-associated protein was phosphorylated in vivo and in vitro by a nuclear serine/threonine kinase(s) which, in a number of different cell lines, appeared to be stimulated in response to interleukin-1 alpha and lipopolysaccharide treatment . In the B cell lines 70Z/3 and CH12 LX2B, and the T cell line EL-4 6.1 C10 the activity of the kappa B-associated kinase(s) correlated with the binding activity of nuclear NF-kappa B displayed in a gel shift assay . In vitro, the 65-kDa protein was phosphorylated in the absence of exogenously added kinase . The 65-kDa phosphoprotein and the kinase activity remained associated following sequential anion-exchange and hydrophobic interaction chromatography . These results suggest that the kappa B-associated phosphoprotein is either autophosphorylated or is phosphorylated by a closely associated kinase(s) . Stimulation of a nuclear protein kinase which is closely associated with a sequence-specific DNA may reflect a novel mechanism by which growth factors regulate gene expression. J Mol Biol, 1991 Jul 5, 220(1), 5 - 7 Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus; Williams RL et al.; Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate . Human and rodent forms of this enzyme have been shown to be suppressors of metastasis . Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli . Two crystal forms have been obtained . Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II . Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis . Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II. J Biol Chem, 1991 Jul 5, 266(19), 12289 - 93 Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin . Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation; Wen ZL et al.; We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 8258-8262) . While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult . We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH . We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor. J Biol Chem, 1991 Jul 5, 266(19), 12140 - 5 Molybdenum cofactor biosynthesis in Escherichia coli . Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide; Johnson JL et al.; The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor . The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin . The molybdenum cofactor in a number of E . coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain . In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E . coli . Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E . coli cells . Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide. Biochemistry, 1991 Jul 2, 30(26), 6454 - 64 Selectivity of Escherichia coli RNA polymerase for template conformation; Butzow JJ et al.; Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself . Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template . Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors of Mg2+ and {Co(NH3)6}3+ . RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by {Co(NH3)6}3+ concentrations much higher than those required for B-Z transition . However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer . When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage . The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation . When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited . Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently . Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer . Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion . These effects were observed for poly(dGdm5C).poly(dGdm5C), with Z stabilized by {Co(NH3)6}3+ or Mg2+ . (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by {Co(NH3)6}3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1991 Jul 2, 30(26), 6484 - 90 Open conformation of a substrate-binding cleft: 19F NMR studies of cleft angle in the D-galactose chemosensory receptor; Luck LA et al.; The Escherichia coli D-galactose and D-glucose receptor is a two-domain structure with a sugar-binding site at the interface between domains . The structure of the closed cleft containing bound D-glucose has been determined crystallographically, but the open cleft remains to be characterized . The present study illustrates a generalizable approach that is used to detect and analyze both the open- and closed-cleft conformations in solution . A 19F nucleus located inside the cleft is monitored by 19F NMR . When the cleft is occupied by D-glucose, the 19F nucleus is found to be inaccessible to the aqueous paramagnetic probe Gd-EDTA, verifying that the occupied cleft is closed in solution and inaccessible to bulk solvent . When the cleft is empty, the 19F nucleus becomes accessible to the paramagnet such that the distance of closest approach is r less than or equal to 10 A, indicating that the empty cleft opens at least transiently by an angle theta greater than or equal to 18 +/- 3 degrees. Biochemistry, 1991 Jul 2, 30(26), 6476 - 83 Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form; Marcy AI et al.; Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components . A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity . Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein . Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values . Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor . Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor . Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies. Biochemistry, 1991 Jul 2, 30(26), 6416 - 22 Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis; Thielking V et al.; Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV . We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity . In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme . Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions . It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues . Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI . On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner. Biochemistry, 1991 Jul 2, 30(26), 6412 - 6 A novel alpha-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase; Williams JS et al.; The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide . The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration . The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively . As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids . In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected . The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group . These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction . The E . coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein {Posorske, L . H., Cohn, M., Yanagisawa, N., & Auld, D . S . (1979) Biochim . Biophys . Acta 576, 128} . On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton . The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS) Carcinogenesis, 1991 Jul, 12(7), 1241 - 6 The reaction of pristane (2,6,10,14-tetramethylpentadecane) with radiolytically generated reactive oxygen intermediates results in a stable genotoxic compound as assessed by the SOS chromotest; Janz S et al.; The most widely studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p . injections of the isoalkane pristane (2,6,10,14-tetramethylpentadecane) . Employing a simple quantitative and well-established short-term bacterial genotoxicity assay, the SOS chromotest, as a model system, we have investigated whether pristane may potentially be involved in causing or modulating the genotoxic events thought to induce plasma cell tumorigenesis . We found that incorporation of pristane into the cell membranes enhance the SOS response in Escherichia coli PQ37 and PQ300 induced by gamma-radiation under hyperoxic conditions . Moreover, the oxidation of pristane by radiolytically generated reactive oxygen intermediates yielded a stable, genotoxic product active on E . coli PQ300, a SOS tester strain designed to detect oxidative genotoxins . We discuss these findings in relation to the tumor-promoting role of the chronic i.p . inflammation that accompanies plasmacytomagenesis and conclude that, under these specific conditions, pristane may possess a previously unrecognized genotoxic activity in its tumorigenic potential. Mutat Res, 1991 Jul, 255(1), 89 - 93 Theoretical and experimental measures of DNA helix stability and their relation to sequence specific repair of O6-ethylguanine lesions; Foley CK et al.; Recent work (Breslauer et al . (1986) Proc . Natl . Acad . Sci . (U.S.A.), 83, 3746) has provided a method for calculating empirical thermodynamic quantities for helix to coil transitions from the base sequence of any oligomer . It is shown in this work that the DNA helix binding energy, calculated with the AMBER force field, for 9-mers of the type 5'-GGGXGeYGGG-3', where X and Y are any base and the central Ge is O6-ethylguanine, correlates well with the empirical delta G for helix to strand transitions . The mutation spectrum of ethane methylsulfonate (EMS) in the lacI gene of Escherichia coli can be modeled using the calculated local binding energy but the empirical free energies, enthalpies and melting temperatures predict these levels of repair less well . The relation of the binding energy to the mutation spectrum can be somewhat improved by including entropic effects in a theoretical free energy of binding as given by delta G theoretical identical to delta E binding - T delta S. Mutat Res, 1991 Jul, 255(1), 39 - 48 Survival of M13mp18 gapped duplex DNA as a function of gap length; Hartke A et al.; Gaps of various lengths were generated in duplex M13mp18DNA by exonuclease III digestion of nicked DNA . The length of the gap increased essentially linearly with time of digestion . Survival in E . coli, however, was not a linear function of gap length . Similar results were obtained when gaps were produced by stopping the polymerization reaction . The survival (N/No) of the gapped DNA in SOS-induced E . coli cells transformed by electroporation and uninduced cells transformed by the calcium chloride method can be quantitatively accounted for by a kinetic model assuming a single-strand endonucleolytic activity (Pd) in the cell which increases linearly with gap length (L) and a repair activity by a polymerase (Pr) which is independent of gap length (formula 1) . With uninduced cells transformed by electroporation the results can be mathematically described if assumptions are made concerning the protection of single-stranded parts of the DNA by single-strand affinic proteins. Mutat Res, 1991 Jul, 249(1), 19 - 27 Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA; Bennett CB et al.; In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated . This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background . On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected . DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes . A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine. Mutat Res, 1991 Jul, 249(1), 177 - 87 Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV; Little CA et al.; Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV) . Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501 . However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested . Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains . Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival . Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect . Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect . These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions . They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions. J Surg Res, 1991 Jul, 51(1), 46 - 53 Adaptive regulation of alanine transport in hepatic plasma membrane vesicles from the endotoxin-treated rat; Pacitti AJ et al.; The mechanisms by which hepatic alanine consumption is increased during endotoxemia were investigated to gain further insight into the altered amino acid metabolism which characterizes critical illness . Rats were studied 12 hr after receiving endotoxin (ENDO) or saline . Hepatic alanine delivery was determined in vivo and hepatic alanine content was measured . Hepatocyte transport activity was studied by evaluation of {3H}-alanine accumulation in hepatocyte plasma membrane vesicles (HPMVs) . Vesicle integrity was demonstrated by electron microscopy and a 14-fold enrichment in 5'-nucleotidase . Endotoxin treatment resulted in a state of hyperalaninemia and a threefold increase in hepatic alanine delivery (2.79 +/- 0.17 mu mole/100 g body weight/min in controls vs 8.13 +/- 0.98 in ENDO animals; P less than 0.001) . Data from HPMVs revealed the presence of a high- and low-affinity component of alanine transport . Endotoxin treatment resulted in a 30% decrease in the Vmax of the high-affinity transport component (3355 +/- 177 pmole/mg protein/10 sec in controls vs 2338 +/- 270 in the ENDO group; P less than 0.05) . Concomitant with the observed changes in alanine delivery and transport activity, endotoxin treatment resulted in a 56% rise in hepatic alanine content (2.53 +/- 0.29 mu mole/g liver in controls vs 3.95 +/- 0.23 in ENDO; P less than 0.005) . These data indicate that the accelerated hepatic alanine consumption which occurs during endotoxemia is primarily the result of increased hepatic substrate delivery . Despite the resultant repression of transport activity, delivery begins to outdistance the metabolic capacity of the hepatocyte to utilize alanine and intracellular alanine levels rise. J Med Chem, 1991 Jul, 34(7), 2094 - 101 Stereospecific synthesis of (R)- and (S)-S-adenosyl-1,8-diamino-3-thiooctane, a potent inhibitor of polyamine biosynthesis . Comparison of asymmetric induction vs enantiomeric synthesis; Liu C et al.; Two diastereomers of the potent spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thiooctane have been prepared in high (greater than 96% de) stereochemical purity . Two synthetic routes were investigated, one based on asymmetric induction and the other involving an enantiomeric synthesis . The latter route gave the desired products in greater than 96% de, whereas the synthesis based on asymmetric induction resulted in only 80% de in the final product . Evaluation of the two diastereomers as inhibitors of spermidine synthase showed that the R diastereomer is a more potent inhibitor than the S diastereomer. J Med Chem, 1991 Jul, 34(7), 1969 - 74 Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages, and adjuvanticity in vivo and in vitro; Metzger J et al.; Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution . 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom) . The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 {2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes . The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity . (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants . A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL . Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone . Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages. J Bacteriol, 1991 Jul, 173(14), 4537 - 9 The hemimethylated replication origin of Escherichia coli can be initiated in vitro; Boye E; Unmethylated, fully methylated, and hemimethylated oriC-containing plasmids were assayed as substrates for DNA replication in vitro by using a system reconstituted with pure proteins . In contrast to the in vivo situation, all three substrates were initiated efficiently; the fully methylated plasmid was about twice as active as the other two. J Bacteriol, 1991 Jul, 173(14), 4424 - 32 Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12; Egan SM et al.; The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression . We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation . We selected and analyzed seven nitrate-independent narL mutants . Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala . The other four mutants were weakly constitutive . The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation . We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles . The first, narL503(H15L), was a recessive null allele . The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity . We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn . This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation . Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain . Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate . This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI. J Bacteriol, 1991 Jul, 173(14), 4404 - 10 Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli; Meury J et al.; Escherichia coli can adapt and recover growth at high osmolarity . Adaptation requires the deplasmolysis of cells previously plasmolyzed by the fast efflux of water promoted by osmotic upshift . Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+ . The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high . The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity . The temperature-sensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E . coli at 30 degrees C. J Bacteriol, 1991 Jul, 173(14), 4288 - 96 Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis; Stibitz S et al.; The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded . Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane . We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins . PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting . Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems . We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS. J Bacteriol, 1991 Jul, 173(14), 4254 - 62 ClpB is the Escherichia coli heat shock protein F84.1; Squires CL et al.; ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease . It has recently been shown that ClpP is a heat shock protein . Here we show that ClpB is the Escherichia coli heat shock protein F84.1 . The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation . Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C . We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C . We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels . This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA . F68.5 can be observed on many published two-dimensional gels of heat-induced E . coli proteins, but the original catalog of 17 heat shock proteins did not include this spot. Eur J Biochem, 1991 Jul 1, 199(1), 47 - 51 Properties of recombinant NADP-malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells; Jacquot JP et al.; In this study, a cDNA clone coding for sorghum leaf NADP-malate dehydrogenase {Cretin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J . C . & Pernollet, J . C . (1990) Eur . J . Biochem . 192, 299-303} was used either in the full-length form or in a shorter form deprived of the 5' end coding for the transit peptide . Both cDNA fragments were cloned into the expression vector pKK233-2 and the resulting constructions were used to transform E . coli cells . The bacterial cells which do not contain any NADP-dependent malate dehydrogenase before transformation were able to express the protein after transformation and induction, as detected both by activity measurements and by immunoblot . The recombinant proteins could be purified to homogeneity and their biochemical characteristics studied . They were identical to those of the enzyme isolated from corn or sorghum leaves, including the well known redox regulatory properties . The NADP-malate dehydrogenases derived from both constructions had a similar subunit size and the analysis of their N-terminal sequences revealed that E . coli cells were able to recognize the processing signal of the precursor polypeptide and to mature and assemble the protein in a manner similar to higher plants. Biochim Biophys Acta, 1991 Jul 1, 1066(1), 21 - 8 Escherichia coli membranes during electrotransformation: an electron microscopy study; Sabelnikov AG et al.; Structural changes undergone by Escherichia coli cell envelope membranes under the conditions of electrically induced gene (DNA) transfer (exponential pulse of about 13 kV/cm, tau = 5 ms) were studied by freeze-fracture electron microscopy . Special device similar to that of Stenger and Hui {1986) J . Membr . Biol . 93, 43-53), that allowed cryofixation of samples almost simultaneously with application of electric pulse, was employed to examine the cells within a short time (less than or equal to 1 s) after the pulse . Extensive blebbing of cells was observed immediately after the pulse . At later times (30-40 s after the pulse) blebbing was not detected, instead infrequent cellular membrane fusion and formation of large membrane 'opening' or pores were observed . An attempt to relate the observed membrane changes with cellular viability and permeability to exogenous DNA failed . Challenge of cells with a plasmid DNA 10 s after the pulse application resulted in a dramatic loss (at least four orders of magnitude) of the number of transformants compared to cells pulsed in the presence of DNA . On the other hand the results on additional pulsing of cell prior to the main electrotransformation procedure suggested that the life-time of membrane defects is at least no less than 2 min . Possible ways to reconcile the results are suggested. Am Rev Respir Dis, 1991 Jul, 144(1), 173 - 8 Lipopolysaccharide-induced pulmonary vascular sequestration of polymorphonuclear leukocytes is complement independent; Cardozo C et al.; Lipopolysaccharide (LPS) injected intravenously produces leukopenia and sequestration of polymorphonuclear leukocytes (PMN) in the pulmonary vascular bed . To evaluate the role of complement in this process, we used C5-sufficient (B10.D2/nSn) and C5-deficient (B10.D2/oSn) mice and Sprague-Dawley rats depleted of complement with Naja naja cobra venom factor (CVF) . We found a comparable increase in the number of PMN in lung tissue of C5-sufficient and C5-deficient mice given Escherichia coli LPS (0127:B8, 3 mg/kg), revealing that LPS acts independently of C5 and its biologically active fragments . Intravenous injection of LPS (3 mg/kg) into rats caused significant intravascular complement activation as assessed by serum CH50 and resulted in an almost 10-fold increase in numbers of PMN in lung tissue . Pretreatment of rats with CVF (50 U) did not reduce LPS-induced PMN sequestration, suggesting that the process is independent of C3 . As reported previously, we found large numbers of PMN in bronchoalveolar lavage samples of 24 h after injection of LPS (3 mg/kg) . Complement depletion did not prevent LPS-induced migration of PMN . No PMN migration occurred 2, 6, 12, 24, or 48 h after injection of CVF alone, indicating that complement activation is not sufficient to cause PMN migration . In contrast to our findings in rats, no PMN migrated into airspaces of C5-sufficient and C5-deficient mice 24 or 48 h after injection of LPS (3 to 20 mg/kg). Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5882 - 6 Adaptive evolution that requires multiple spontaneous mutations: mutations involving base substitutions; Hall BG; A previous study has demonstrated that adaptive missense mutations occur in the trp operon of Escherichia coli . In this study it is shown that, under conditions of intense selection, a strain carrying missense mutations in both trpA and trpB reverts to Trp+ 10(8) times more frequently than would be expected if the two mutations were the result of independent events . Comparison of the single mutation rates with the double mutation rate and information obtained by sequencing DNA from double revertants show that neither our classical understanding of spontaneous mutation processes nor extant models for adaptive mutations can account for all of the observations . Despite a current lack of mechanistic understanding, it is clear that adaptive mutations can permit advantageous phenotypes that require multiple mutations to arise and that they appear enormously more frequently than would be expected. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5799 - 803 Conformational and membrane-binding properties of a signal sequence are largely unaltered by its adjacent mature region; McKnight CJ et al.; We have synthesized a peptide corresponding to the 25-residue signal sequence plus the first 28 residues of the Escherichia coli outer membrane protein LamB in order to explore the properties of a signal sequence in the presence of the N-terminal region of its passenger . In the last few years, there have been several observations of differing efficiencies of export when signal sequences are attached to different passenger proteins or when the first part of a passenger protein undergoes mutation . In the LamB case, gene fusions with lacZ have shown that the signal sequence plus the first 28 residues of mature LamB are necessary to direct beta-galactosidase into the export pathway {Rasmussen, B . A . & Silhavy, T . J . (1987) Genes Dev . 1, 185-196} . The origin of these observations and whether there is an influence of the mature region on the properties of the signal sequence have not been known . We find that the conformational and membrane-binding properties of the LamB signal sequence manifest in a 25-residue peptide are essentially unaltered in the context of the 53-residue peptide corresponding to this signal sequence plus the first 28 residues of the mature LamB protein . CD spectra show that the signal peptide and passenger domains are conformationally independent of each other in micelle or bilayer environments . Furthermore, the signal sequence leads to the spontaneous association of the 53-residue peptide with a lipid bilayer; alone, the mature domain does not interact with lipid bilayers . Fluorescence results show that the mode of interaction of the signal peptide with a bilayer is essentially unaltered by the presence of its mature region . This lack of influence of the mature domain on the behavior of the signal sequence is unexpected for juxtaposed polypeptides of comparable length and may be of physiological importance: N-terminal regions of secreted proteins may be selected to be passive, by comparison with their cognate signal sequences, which themselves must engage the export apparatus and actively interact with its components. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5729 - 33 A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans; Maruyama IN et al.; Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans . Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C . The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner . These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system. J Bacteriol, 1991 Jul, 173(13), 4188 - 94 The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli; McCann MP et al.; KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex . Mutants with the katF gene survive carbon and nitrogen starvation poorly . Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses . Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature . Thus, KatF is a major switch that controls the starvation-mediated resistant state in E . coli. J Bacteriol, 1991 Jul, 173(13), 4133 - 43 Regulation of expression of the Escherichia coli K-12 mtr gene by TyrR protein and Trp repressor; Sarsero JP et al.; The Escherichia coli K-12 mtr gene, which encodes a tryptophan-specific permease, was cloned, and its nucleotide sequence was determined . The precise location of the mtr gene at 69 min on the E . coli chromosome was determined . The mtr gene product was identified as a 414-amino-acid residue protein with a calculated molecular weight of 44,332 . The protein is very hydrophobic, consistent with its presumed location spanning the cytoplasmic membrane . The initiation sites of transcription and translation were identified . Construction of an mtr-lacZ transcriptional fusion facilitated investigation of the molecular basis of mtr regulation . The TyrR protein in association with phenylalanine or tyrosine is responsible for the activation of mtr expression, whereas the Trp repressor in conjunction with tryptophan serves to repress expression of this gene . Site-directed mutagenesis confirmed that sequences in the mtr regulatory region homologous to TyrR protein and to Trp repressor-binding sites were involved in the activation and repression of mtr expression, respectively . Sequences homologous to sigma 70- and sigma 54-dependent promoters were identified upstream of the transcription start point of mtr . It was determined that transcription of mtr occurs only via a sigma 70-dependent promoter. J Bacteriol, 1991 Jul, 173(13), 4049 - 55 Mutant MotB proteins in Escherichia coli; Blair DF et al.; The MotB protein of Escherichia coli is an essential component in each of eight torque generators in the flagellar rotary motor . Based on its membrane topology, it has been suggested that MotB might be a linker that fastens the torque-generating machinery to the cell wall . Here, we report the isolation and characterization of a number of motB mutants . As found previously for motA, many alleles of motB were dominant, as expected if MotB is a component of the motor . In other respects, however, the motB mutants differed from the motA mutants . Most of the mutations mapped to a hydrophilic, periplasmic domain of the protein, and nothing comparable to the slow-swimming alleles of motA, which show normal torque when tethered, was found . Some motB mutants retained partial function, but when tethered they produced subnormal torque, indicating that their motors contained only one or two functional torque generators . These results support the hypothesis that MotB is a linker. J Leukoc Biol, 1991 Jul, 50(1), 77 - 85 Effect of chronic endotoxemia on concanavalin A-stimulated inositol lipid metabolism in rat splenocytes; Hagar AF et al.; The effect of a chronic, nonlethal infusion of endotoxin (ET) on the phosphatidylinositol (PI) cycle in rat splenocytes was evaluated . Rats were infused intravenously with sterile saline or Escherichia coli ET (0.1 mg/100 g body weight/24 hr) for 30 hr via subcutaneously implanted osmotic pumps . The splenocytes were then labeled in vitro for 90 min with {32P}PO4 or for 3 hr with {3H}myoinositol to assess the status of the resynthesis and degradative parts of the PI cycle, respectively . PI cycle activity was depressed in splenocytes of ET-infused rats as evidenced by a 25% reduction in the incorporation of {32P}PO4 into phosphatidic acid (PA), PI, and the polyphosphoinositides and by a similar decrease in the production of {3H}inositol phosphates . Stimulation of splenocytes with concanavalin A (Con A) resulted in dose-dependent increases in the incorporation of {32P}PO4 into PA and PI and in the production of {3H}inositol phosphates, indicating that Con A stimulates the PI cycle in these cells . The Con A-stimulated increase in inositol phosphate production was higher in splenocytes from ET-infused rats . We have previously shown that splenocytes from rats infused for 30 hr with ET exhibit a decreased blastogenic responsiveness to Con A and lipopolysaccharide {Spitzer et al., Proc . Soc . Exp . Biol . Med . 186,27, 1987} . The present data do not support the notion that inositol lipid-mediated signaling mechanisms are solely responsible for the expression of the appropriate functional response and suggest that in ET-infused rats there is an uncoupling of the initial response to Con A (i.e., the production of inositol lipid-derived second messengers) and the long-term (i.e., mitogenic) response. J Clin Invest, 1991 Jul, 88(1), 93 - 100 Mechanism of mammalian cell lysis mediated by peptide defensins . Evidence for an initial alteration of the plasma membrane; Lichtenstein A; Defensins induce ion channels in model lipid bilayers and permeabilize the membranes of Escherichia coli . We investigated whether similar membrane-active events occur during defensin-mediated cytolysis of tumor cells . Although defensin-treated K562 targets did not release chromium-labeled cytoplasmic components for 5-6 h, they experienced a rapid collapse (within minutes) of the membrane potential, efflux of rubidium, and influx of trypan blue . Defensin treatment also blunted the subsequent acidification response induced by nigericin, thereby further supporting the notion of enhanced transmembrane ion flow during exposure . These initial effects on the plasma membrane were not sufficient for subsequent lysis; a second phase of injury was required which involved the continued presence of defensin . The rapid membrane permeabilization phase was inhibited by azide/2-deoxyglucose, cytochalasin B, and increased concentrations of extracellular potassium and was unaffected by actinomycin-D, cycloheximide, and varying the calcium concentration . In contrast, the second phase was unaffected by cytochalasin B, inhibited by azide/2-deoxyglucose, enhanced by actinomycin D and cycloheximide, and varied with calcium concentration . These results indicate the initial adverse effect of defensins on mammalian cells occurs at the cell membrane . It is possible that the second phase of injury is mediated intracellularly by defensin that has been internalized through this leaky membrane. Crit Care Med, 1991 Jul, 19(7), 955 - 62 Lung fluid dynamics and supply dependency of oxygen uptake during experimental endotoxic shock and volume resuscitation; D'Orio V et al.; BACKGROUND AND METHODS: We studied the effect of volume resuscitation on lung fluid balance and systemic oxygen extraction during septic shock in eight anesthetized dogs . Sepsis was induced using a 2-hr continuous infusion of Escherichia coli endotoxin at 0.25 micrograms/min.kg . Relationships between oxygen uptake (VO2) and oxygen supply (DO2) were performed acutely during stepwise controlled decrements in cardiac output by progressive inflation of an intracardiac balloon . At each stage, DO2 and corresponding VO2 were measured independently and the individual critical DO2 level was referred to as the point below which the relationship held . The slope of such a constructed relationship was defined as the maximal oxygen extraction ratio . Lung fluid balance was assessed by measurements of extravascular lung water . All values were studied at baseline, after endotoxin insult, and after reversing hypotension by a 10% dextran infusion . RESULTS: Endotoxin infusion led to a shock state that associated hypotension (from 135 to 63 mm Hg) with increases in blood lactate (from 0.53 to 3.9 mmol/L) . The mean critical DO2 and maximal oxygen extraction ratio were significantly altered from 7.9 to 17.8 mL/min.kg and from 0.81 to 0.38, respectively . After reversing hypotension by 28 mL/kg colloid infusion, the critical DO2 (11.4 mL/min.kg) and maximal oxygen extraction ratio (0.48) were significantly improved . However, restoration of normal values required a state of fluid overload by further dextran infusion (8 mL/kg) . At the end of the fluid challenge, extravascular lung water significantly increased from 6.4 to 17.4 mL/kg . CONCLUSIONS: These data suggest that volume loading may reverse endotoxin-induced peripheral perfusion abnormalities . However, substantial pulmonary edema may occur, possibly jeopardizing the beneficial effects of fluid expansion. Virology, 1991 Jul, 183(1), 151 - 9 Biochemical and biological comparison of HIV-1 NEF and ras gene products; Nebreda AR et al.; Human immunodeficiency virus type 1 (HIV-1) NEF protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or rats . To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia coli using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins . ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties . In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP . This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position . Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes . Also, unlike ras, the NEF gene products failed to induce meiotic maturation when injected into fully grown Xenopus oocytes. J Immunol, 1991 Jul 1, 147(1), 312 - 9 Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae; McKenzie KR et al.; The gene encoding the Mycobacterium leprae 70-kDa heat shock protein has been isolated from a cosmid library using a fragment of the clone JKL2 . Southern blot analysis of a positive clone identified a 4.4-kb fragment containing the entire coding region of the gene plus 2.4 kb upstream . Sequencing revealed the gene to encode a 621-amino acid protein, bearing 56% identity with the Escherichia coli dnaK gene product and 47% and 46% identity with the human and Caenorhabditis elegans hsp70, respectively . Comparison with the C-terminal 203 amino acids of the Mycobacterium tuberculosis 71-kDa Ag yielded 70% identity . Recombinant M . leprae p70 was produced in E . coli as a fusion protein (rp70f) with a portion of the schistosomal glutathione-S-transferase, using the expression vector, pGEX-2T . Cleavage with thrombin resulted in the release of a 70.0-kDa protein (rp70c) from the glutathione-S-transferase . Examination of the proteins by immunoblotting demonstrated that anti-M . leprae mAb, L7, and sera from lepromatous leprosy patients bound to both the cleaved and fusion proteins . We compared the T cell reactivity of the M . leprae recombinant proteins with that of mAb affinity-purified bacille Calmette-Guerin (BCG) 70-kDa Ag using proliferation assays . PBMC of BCG vaccinees responded to both M . leprae cleaved and fusion p70, though more subjects responded to the rp70c (18 of 20) than to rp70f (13 of 20) . Responses were generally higher to rp70c than to rp70f, however all responses to the M . leprae recombinant proteins were lower than to mAb affinity-purified BCG p70 . Thus, the M . leprae 70-kDa heat shock protein elicits T and B cell responses in subjects exposed to mycobacteria, despite its homology with the human hsp70. J Cell Biol, 1991 Jul, 114(1), 83 - 99 Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil; Wiche G et al.; We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones . The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains . The plectin sequence has a number of repeating motifs . The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules . The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif . The plectin sequence has several marked similarities to that of desmoplakin (Green, K . J., D . A . D . Parry, P . M . Steinert, M . L . A . Virata, R . M . Wagner, B . D . Angst, and L.A . Nilles . 1990 . J . Biol . Chem . 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin . The plectin sequence also has homologies to that of the bullous pemphigoid antigen . Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution . There appeared to be a single rat plectin gene that gave rise to a 15-kb message . Expression of polypeptides encoded by defined fragments of plectin cDNA in E . coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies . This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain. EMBO J, 1991 Jul, 10(7), 1853 - 62 TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III; Simmen KA et al.; We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene . Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays . First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract . Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates . Third, TFIID can replace one of two heat labile fractions essential for U6 transcription . Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases. J Virol, 1991 Jul, 65(7), 3715 - 20 A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene; Wright CF et al.; A factor, designated VLTF-1, that is required in vitro for specific transcription of vaccinia virus late genes was previously isolated from vaccinia virus-infected cells . Subsequent genetic experiments identified three vaccinia virus genes, encoding proteins of 17, 26, and 30 kDa, that together trans activate late gene expression in vivo . The purpose of this study was to determine whether VLTF-1 corresponded to one of the three trans activators . Toward this end, VLTF-1 was further purified, the trans-activator genes were expressed in Escherichia coli, and antisera were made to the native and recombinant proteins . Antibody to the 30-kDa recombinant protein reacted on Western immunoblots with a protein of approximately Mr 30,000 that cochromatographed and cosedimented with VLTF-1 activity from virus-infected cells . Conversely, antibody to purified VLTF-1 bound to products produced by in vitro transcription and translation of the open reading frame encoding the 30-kDa trans-activator protein . Both antisera depleted VLTF-1 activity and blocked late gene transcription by partially purified extracts of vaccinia virus-infected cells . Taken together, these data demonstrate that the 30-kDa trans activator comprises part, if not all, of VLTF-1 activity. Res Microbiol, 1991 Jul-Aug, 142(6), 623 - 31 Isolation and characterization of intragenic suppressors of an Escherichia coli ftsA mutation; Robinson AC et al.; Mutagenesis of a strain of Escherichia coli carrying a temperature-sensitive (Ts) mutation in the cell division gene ftsA yielded a number of temperature-resistant variants . In certain cases, restoration of viability at the restrictive temperature could not be attributed to suppressor mutations occurring in other genes or to structural gene reversion . DNA sequencing of the variants demonstrated the continuing presence of the original Ts mutation (ftsA13) and revealed secondary mutations within the same gene . These secondary mutations are able to rescue the ftsA13 mutation in cis, but not in trans. J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1529 - 36 Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein; Mitchison M et al.; A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L . interrogans) serovar hardjo was cloned and expressed in Escherichia coli . Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18 . Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa . The G + C content of the flaB gene was 54.7 mol% . Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins. Biotechniques, 1991 Jul, 11(1), 18, 20, 22 - 4 Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction; Cole KD; A method for the rapid preparation of DNA is described . The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system . A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs . Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region . Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample . The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion . This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins . The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents . With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase . The paper describes a method for DNA isolation that is rapid, simple and economical. Biotechniques, 1991 Jul, 11(1), 14 - 6 Chemiluminescent substrates increase sensitivity of antigen detection in western blots; Sandhu GS et al.; Replacement of colorimetric substrates in Western blots by chemiluminescent reagents enhances the sensitivity of detection by more than an order of magnitude . Protein levels beyond the detection limit of colorimetric substrates can be consistently detected without modifying pre-existing laboratory protocols. Zentralbl Veterinarmed A, 1991 Jul, 38(6), 445 - 59 Role of eicosanoids in abortion and its prevention by treatment with flunixin meglumine in cows during the first trimester of pregnancy; Giri SN et al.; Intravenous infusion of E . coli endotoxin at a rate of 4.16 ng/kg/min over 6 hr (total dose 1.5 micrograms/kg) in 5 cows in the first trimester of gestation induced abortion between 60 and 72 hr in three cows . Plasma PGF2 alpha levels in the aborting cows increased significantly to 289% of the zero time control (ZTC) at 1 hr and remained elevated for 9 hr . The PGF2 alpha level remained unaffected in the non-aborting cows except at 2 hr . The plasma TxB2 levels were increased by 6 to 18 fold for 6 hr in both the aborting and non-aborting cows relative to their ZTC controls . The 6-keto-PGF1 alpha levels were significantly increased to 2 to 3 fold only in the aborting cows . Plasma cortisol levels were increased maximally to 1,500% of ZTC at 5 hr in the aborting cows . Thereafter, the levels gradually declined but remained significantly elevated for 24 hr . The increases in the cortisol levels in the non-aborting cows were only 280% of ZTC at 5 hr and returned to ZTC value by 12 hr . Plasma progesterone levels in the aborting cows remained unaffected until 12 hr followed by a progressive decline through 18 hr to extremely low levels at 3, 4, and 5 days . Endotoxin-infusion caused hyperglycemia in both aborting and non-aborting cows and lactic acidemia in the aborting cows . Treatment with two doses of flunixin meglumine (FM, 1.1 mg/kg), an inhibitor of cyclooxygenase, 1 hr prior to endotoxin infusion and then 13 hr later, completely prevented the endotoxin-induced abortion and increases in the plasma PGF2 alpha, TxB2 and 6-keto-PGF1 alpha concentrations . The PGE level remained unaffected . Although FM treatment failed to abolish endotoxin-induced increases in the plasma cortisol and lactic acid levels, it effectively prevented marked decreases in the progesterone and increases in the glucose concentrations . It was concluded that the use of FM offers therapeutic promise in preventing bovine abortion caused by endotoxin resulting from bacterial infection during the 1st trimester of gestation. Vet Q, 1991 Jul, 13(3), 155 - 62 The role of endotoxin in the pathogenesis of acute bovine laminitis; Boosman R et al.; To study the possible role of endotoxin in the pathogenesis of bovine laminitis, local and systemic injections of endotoxin (E . coli 0111 B4) with different doses were given to three groups of four cows each . Clinical and haematologic parameters indicated an acute-phase response, including positive plasma ethanol gelation (soluble fibrin), the occurrence of fibrin degradation products and decreased thrombocyte counts . Local Shwartzman reactions were not evoked . Clinical examination of the claws and the gait of the animals revealed no signs of laminitis . However, on histopathological examination of the claw corium signs of laminitis such as vacuolisation of the Stratum basale, lymphocyte and leucocyte infiltration and thrombosis were found . These results indicate that endotoxin indeed may be involved in the pathogenesis of laminitis . For the development of a clinical acute laminitis model in cattle either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed. Mutagenesis, 1991 Jul, 6(4), 271 - 8 Mutagenic activity of aziridinyl steroids and their mechanism of action in biological systems; Islam S et al.; The aziridinyl derivatives of steroids, structurally related to cholesterol, were tested for their mutagenic activity in the Ames tester strains . The test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity . A significant decrease in the survival of SOS defective mutants, recA and lexA of Escherichia coli was observed as compared with their wild-type counterpart in the presence of the steroids . The role of SOS repair genes gains further support from the lambda prophage induction in the lysogen and beta-galactosidase induction in the Mud strains as well as mutagenesis with Ames tester strains . Structural features which appear essential for mutagenic activity in these strains are (i) a reactive N-aminophthalamide group at the (5,6-b) position and (ii) an acetoxy/chlorine group at the third position of the steroidal nucleus . The individual moieties/groups were not mutagenic per se . These steroids appear to generate H2O2 as well as superoxide and hydroxyl radicals in the model biological system. Mol Microbiol, 1991 Jul, 5(7), 1801 - 10 Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell; Miczak A et al.; Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm . The RNA-processing enzymes were associated with the membrane fraction . The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction . By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane . The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity . The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed. Mol Microbiol, 1991 Jul, 5(7), 1791 - 9 Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic processing; Nilsson P et al.; Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities . As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript . Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism . The processing did not occur under non-permissive conditions in an E . coli rne mutant strain with a temperature-sensitive RNaseE . The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA . A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites . Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event . However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E . coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency . Our findings are consistent with the idea that ams is related/allelic to rne . Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript . We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1991 Jul, 5(7), 1779 - 90 Characterization of the oriT region of the IncFV plasmid pED208; Di Laurenzio L et al.; DNA sequence analysis of a 2.2kb EcoRI-HindIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasmids . The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm . Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region . These three footprint regions contained a Hinfl-like sequence (GANTC) that appeared 16 times, spaced 11-12 bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region. Mol Microbiol, 1991 Jul, 5(7), 1681 - 6 Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation; Pla J et al.; An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid . Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival . Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell . Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm. Mol Microbiol, 1991 Jul, 5(7), 1639 - 48 The gef gene from Escherichia coli is regulated at the level of translation; Poulsen LK et al.; We describe post-transcriptional regulation of the chromosomal gene, gef, from Escherichia coli . The gef gene is a member of a gene family consisting of the chromosomal gef and relF genes from Escherichia coli and the hok, flmA, srnB, and pndA genes, which are situated on conjugative plasmids . All the genes encode small, toxic proteins of approximately 50 amino acids which are functionally and structurally homologous . Furthermore, the gene family shares post-transcriptional regulation of expression, albeit by different mechanisms . We demonstrate here that translation of gef is coupled to an upstream open reading frame which, in turn, is regulated by a transacting factor, probably an antisense RNA. Mol Microbiol, 1991 Jul, 5(7), 1627 - 37 Topographic analysis of the toxic Gef protein from Escherichia coli; Poulsen LK et al.; The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids . We demonstrate here that the Gef protein is detectable by anti-peptide antibodies . Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N-terminal part of the protein, and that the C-terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond . By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect. Mol Microbiol, 1991 Jul, 5(7), 1607 - 13 Filamentous phage assembly; Russel M; Filamentous phages present a genetically well-defined system for studying the ordered membrane assembly of five different phage-encoded proteins around the circular single-stranded DNA phage genome . Assembly occurs at high efficiency in vivo, catalysed by two phage-encoded membrane proteins and at least one host protein, thioredoxin . This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the role of the three morphogenetic proteins . The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes. Mol Microbiol, 1991 Jul, 5(7), 1585 - 92 TyrR protein of Escherichia coli and its role as repressor and activator; Pittard AJ et al.; The TyrR protein regulates the expression of eight transcriptional units that comprise the TyrR regulon . In all but one case, regulation is by repression, while in two cases activation of expression can occur . Notwithstanding the fact that the TyrR protein contains an ATP-binding domain and a helix-turn-helix DNA-binding domain which are structurally homologous to domains of similar functions in proteins such as NifA, NtrC, DctD and XylR, it differs from them in a number of respects . It is not a part of a two-protein component system and it lacks the amino-terminal domain that is present on NtrC and DctD . It activates transcription from 'E sigma 70, promoters but not from 'E sigma 54, promoters . ATP binding seems to be essential for tyrosine-mediated repression but not for activation . In addition, the activity of the TyrR protein is modulated by the binding of one or more of the aromatic amino acids . The consensus sequence for TyrR-binding sites in DNA, referred to as TyrR boxes, is TGTAAAN6TTTACA . Tyrosine-mediated repression occurs at operators containing a pair of adjacent boxes . These have unequal affinities for the TyrR protein . The box that overlaps the RNA polymerase binding site is only bound by TyrR in the presence of both ATP and tyrosine, and binding appears to involve co-operativity between two TyrR protein dimers . In contrast, activation of expression by TyrR appears to require phenylalanine but not ATP.(ABSTRACT TRUNCATED AT 250 WORDS) Med Trop (Mars), 1991 Jul-Sep, 51(3), 363 - 6 {Non-obstructive necrotizing acute enterocolitis (apropos of a case report) )University Hospital Center Kamenge-Bujumbura)}; Armstrong O et al.; The authors report on a new case of acute necrotic non obstructive enterocolitis . Discussion is mainly centered on the nosological framework of this uncommon affection, occurring in an infectious background, and on its etiopathogenetic mechanism . Several hypothesis are formulated . Because of the seriousness of its prognosis, it is emphasized on the necessity of a rapid surgical intervention. J Auton Nerv Syst, 1991 Jul, 35(1), 53 - 62 Secretory reflexes in ileum and jejunum: absence of remote effects; Hubel KA et al.; We have tested the hypothesis that luminal secretagogues initiate neural reflexes that alter ion transport in small intestinal segments proximal or distal to the site of the secretory stimulus . Effects of secretagogues that act by different mechanisms were studied in vitro by measuring short circuit current (ISC) of ileum or jejunum mounted in a unique flux chamber while proximal mucosa in neural continuity with the tissue was perfused with secretagogues (Na deoxycholate . Escherichia coli STa, 5-hydroxytryptamine, theophylline) or was stimulated electrically (EFS) . No proximal stimulus affected distal ISC . We also studied in vivo adjacent segments of ileum or jejunum in neural continuity but with unconnected lumens . In anesthetized rabbits, we measured transmural electrical potential difference and fluid movement (Phenol red marker) . Stimulation of proximal segments of ileum or jejunum with STa, or of ileum with 5-HT or Na deoxycholate did not affect distal transport . Stimulation of distal segments of ileum and jejunum with STa or 5-HT, or of jejunum with Ha deoxycholate did not affect proximal transport . We conclude that the secretion caused by luminal secretagogues in the rabbit small intestine is limited to the area of stimulation. Int Surg, 1991 Jul-Sep, 76(3), 146 - 8 Acute acalculous cholecystitis; Coelho JC et al.; Thirty-two patients with acute acalculous cholecystitis are presented . The age of the patients ranged from 1 to 80 years, with an average of 46.3 years . Acute acalculous cholecystitis occurred during the postoperative period in only four patients . Three patients were receiving total parenteral nutrition and 16 patients had one or more associated medical diseases . One patient had acute acalculous cholecystitis due to mechanical obstruction of the cystic duct caused by a diaphragmatic hernia . The most frequent signs and symptoms were right upper quadrant abdominal pain, nausea, vomiting, fever, abdominal mass, and jaundice . All patients were subjected to cholecystectomy . Nine (28.1%) gallbladder specimens had gangrene . Pericholecystic perforation was observed in four patients (12.5%) free perforation in one patient (3.1%), and empyema of the gallbladder in one patient (3.1%) . Bacteria were cultured from 18 of 24 bile specimens . E . coli was the most common organism isolated . The overall postoperative mortality and complication rates were 15.6% and 40.6% respectively . The average hospital stay was 16.4 days. Int J Pept Protein Res, 1991 Jul, 38(1), 47 - 53 A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange; Rubinfeld B et al.; Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F . {1989} Nature 340, 678-679) . A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity . At a concentration of 25 microM, p891 inhibited GAP activity approximately 50% . Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP . This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891 . In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891 . The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go . p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891 . Also tested were three ras-related GTP-binding proteins; rap, G25K and rac . The rap.-GDP complex was unaffected by 10 microM p891 . Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively . Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras . In addition, p891 independently affects the nucleotide exchange properties of p21ras. Curr Genet, 1991 Jul, 20(1-2), 1 - 3 Ethanol improves the transformation efficiency of intact yeast cells; Lauermann V; A technique is described in which ethanol is used to improve the genetic transformation of intact yeast (Saccharomyces cerevisiae) cells pretreated with LiAc and PEG . Transformation efficiency was increased with increasing concentrations of ethanol with a peak at 10% concentration . The effect varies with different yeast strains and plasmids and up to a maximum of a 15-fold increase was observed. FEMS Microbiol Rev, 1991 Jul, 8(1), 27 - 41 The biology of colicin M; Harkness RE et al.; This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group . The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified . With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins . The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity . To kill, the colicin must enter from outside the cell . Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid . Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis . Colicin M is the only colicin known to have such a mechanism of action . Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da . This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin . Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins . Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids . The mechanism of colicin M release is not known. Protein Seq Data Anal, 1991 Jul, 4(1), 43 - 7 Identification of regulatory building blocks in Escherichia coli genome; Kunisawa T et al.; The nucleotide sequences of extragenic regions in the Escherichia coli genome are statistically analyzed . Sequence elements with high occurrence frequencies are identified; these elements are: (1) extragenic palindromic sequences, which are markedly distinguishable from the already identified repetitive extragenic palindromic sequences; (2) promoter sequences of purine biosynthetic genes; and (3) rho-independent terminator sequences . The repetitious occurrence and extensive sequence similarities suggest that these elements share common evolutionary origins . Copies of one sequence element would have become distributed to various positions on the genome during evolution and have been fixed at locations that provide a selective advantage . The extragenic regions of the E . coli genome seem to consist of various regulatory 'building blocks', similar to a protein which consists of modules or domains. Nucl Med Commun, 1991 Jul, 12(7), 637 - 44 Comparison of 99Tcm-labelled monoclonal anti-granulocyte antibody and 111In-labelled IgG for the detection of focal sites of infection in rats; Juweid M et al.; The abilities of 99Tcm-labelled monoclonal anti-human granulocyte antibody (AGAb) and 111In-labelled nonspecific polyclonal human immunoglobulin (IgG) to localize at focal sites of inflammation were compared in rats with deep thigh infection due to E . coli . The radiolabelled antibodies were coadministered followed 4-6 and 24 h later by imaging and biodistribution studies . At 4-6 h after injection, the target to background ratio (T/B, lesion to contralateral leg) and percentage residual activity (% RA, counts in the lesion/total body counts) were nearly identical for both antibody preparations . At 24 h, T/B and % RA increased significantly (P less than 0.001) for both proteins but differences between the agents were not significant . In vitro analysis of the binding of AGAb and human polyclonal IgG to rat granulocytes showed a low level of binding with both agents . These results suggest that the primary mechanism of localization, by either antibody preparation in this model, is not antigen related . 111In-labelled nonspecific human IgG and 99Tcm-AGAb are equivalent reagents for the detection of focal sites of infection in the rat. J Assoc Off Anal Chem, 1991 Jul-Aug, 74(4), 635 - 48 Dry rehydratable film for enumeration of total coliforms and Escherichia coli in foods: collaborative study; Curiale MS et al.; Rehydratable dry-film plating methods for total coliforms and Escherichia coli in foods have been compared to the AOAC most probable number methods . Fourteen laboratories participated in the collaborative study . Three coliform and E . coli levels in 6 samples of 4 product types (flour, nuts, cheese, and beef with gravy) and in 3 samples of 2 product types (mushrooms and raw turkey) were tested in duplicate by the participants . The mean log counts for the 3 methods were comparable . In general, the repeatability and reproducibility variances of the plating methods were as good as or better than that of the MPN method . The method has been adopted official first action by AOAC. Jikken Dobutsu, 1991 Jul, 40(3), 357 - 60 Pyrogenic responses of the pika, Ochotona rufescens rufescens; Sakai T et al.; In 3-month-old male and female pikas, Ochotona rufescens rufescens, reared at an ambient temperature of 25 degrees C, the mean within-day body temperature was 39.2 degrees C without significant variance . At an ambient temperature of 35 degrees C it increased markedly in males while decreased slightly at 5 degrees C . The pikas showed lower sensitivity than Japanese white rabbits to a pyrogenic E . coli endotoxin. Equine Vet J, 1991 Jul, 23(4), 303 - 8 Whole blood re-calcification time in equine colic; Henry MM et al.; Whole blood re-calcification times were evaluated as a measure of endotoxin-associated coagulopathy in horses . First, the effects of endotoxin concentration and duration of in vitro incubation of citrated whole blood with endotoxin on the whole blood re-calcification time of blood collected from healthy horses were determined . Increasing concentrations or incubation times of endotoxin accelerated the whole blood re-calcification time . This effect was attributed mainly to increased monocyte thromboplastin activity . Second, whole blood re-calcification time, a clotting profile, plasma factor VII activity and plasma endotoxin concentration on blood samples obtained from 35 equine colic patients and 10 healthy horses were determined . Compared with healthy horses, colic patients had a longer mean whole blood re-calcification and prothrombin time, lower per cent factor VII activity and higher mean fibrin degradation products concentration . Within the colic patient group, horses that did not survive had detectable endotoxin in plasma, longer whole blood re-calcification and prothrombin times, and lower plasma factor VII activity, compared with colic patients that survived . These data indicate that colic patients with endotoxaemia experience hypercoagulable states, followed by consumptive coagulopathy . Although the cause of endotoxin-associated coagulopathy is likely multi-factorial, increased expression of monocyte thromboplastin activity may be involved in the pathogenesis of coagulopathy . The whole blood recalcification time is a simple, fast and inexpensive way to detect coagulopathy during endotoxaemia and determine the prognosis for survival. Can J Microbiol, 1991 Jul, 37(7), 509 - 12 Competition between strains of Escherichia coli with and without plasmid RP4 during chemostat growth; Numan Z et al.; Escherichia coli strains J53(nal) and J53(RP4) were grown together in glucose-limited continuous cultures . Based on the measured growth kinetic constants of the two strains, take-over of the cultures by J53(RP4) was predicted . However, in practice, an initial period of predominance by J53(RP4) was always followed by a prolonged period in which relative numerical proportions of the two strains oscillated widely . This period of oscillation was removed or greatly reduced when the difference between the predicted growth-rate potentials of the two strains was increased by selection of a chemostat-adapted variant of J53(RP4). Comput Appl Biosci, 1991 Jul, 7(3), 301 - 8 Artificial intelligence methods for theory representation and hypothesis formation; Karp PD; This article describes artificial intelligence methods for representing theories in molecular biology, and for improving the predictive power of these theories using experimental data . A program called GENSIM provides a framework for representing theories that includes descriptions of classes of biological objects (genes, enzymes, etc.), and processes that specify potential interactions among these objects (such as enzymatic reactions) . GENSIM can employ a theory specified within this framework to predict the outcomes of biological experiments . A program called HYPGENE comes into play when the observed outcome of an experiment does not match the outcome predicted by GENSIM . HYPGENE works backward from the error in GENSIMs prediction to postulate changes to both the theory embodied by GENSIM, and the presumed initial conditions of the experiment . I view HYPGENEs hypothesis generation task as a design problem, and I have adapted AI methods developed for design and planning to this task . These techniques were developed in conjunction with an in-depth study of the discovery of the gene regulation mechanism of attenuation in the E . coli tryptophan operon . Both GENSIM and HYPGENE have been tested on sample problems from the history of attenuation, and produced many of the same solutions as biologists did. Vet Microbiol, 1991 Jul, 28(2), 189 - 98 Controlling acute Escherichia coli mastitis during the periparturient period with recombinant bovine interferon gamma; Sordillo LM et al.; The efficacy of recombinant bovine interferon (rBoIFN)-gamma against experimentally induced Escherichia coli mastitis during the periparturient period was investigated . Dairy cows intramammarily treated with rBoIFN-gamma 24 h before the E . coli challenge had fewer infected quarters, lower clinical scores, and infections of shorter duration when compared to placebo-treated animals . All rBoIFN-gamma treated cows survived the experimental E . coli challenge . However, placebo treated cows had a 42% mortality rate attributed to coliform mastitis within 3 days of the challenge . Results from this study suggest that intramammary infusion of rBoIFN-gamma can prevent the rapid, unrestricted growth of E . coli within the mammary gland and inhibit the subsequent development of an unlimited inflammatory response under experimental conditions . It is likely that controlling severe local inflammatory reactions may also decrease the pathological alterations to mammary parenchymal tissue that often accompanies acute coliform mastitis during the periparturient period . The potential for prophylactic treatment of perinatal dairy cows with rBoIFN-gamma to regulate the rate, severity, and duration of naturally occurring coliform mastitis during periods of heightened susceptibility is discussed. Oncogene, 1991 Jul, 6(7), 1243 - 50 In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes; Bannister AJ et al.; The leucine zipper family of proteins have a DNA binding domain composed of a leucine zipper dimerisation interface and a basic DNA binding structure . We show here that redox changes affect the in vitro DNA binding ability of a select subset of leucine zipper proteins . The bacterially expressed DNA binding domains of Fos/Jun and BZLF1 are unable to bind DNA under non-reducing conditions whereas binding of the C/EBP DNA binding domain is unaffected . Sensitivity to redox state is due to the presence of a conserved cysteine residue in the basic DNA binding motif of Fos, Jun and BZLF1 but not C/EBP . Under non-reducing conditions an intermolecular disulphide bridge is formed between the cysteine residues of each basic motif within a dimer, which prevents DNA binding . We show that oxidation of these C residues can be achieved enzymatically, using glutathione peroxidase, and that DNA binding protects them from oxidation . These data raise the possibility that intracellular changes in the redox state may differentially regulate the activity of leucine zipper family members . In addition the loss of DNA binding activity under non-reducing conditions has implications for the purification methods used to isolate proteins of the leucine zipper family for structural analysis. J Bacteriol, 1991 Jul, 173(14), 4440 - 6 The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii; Joerger RD et al.; Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK) . However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase . Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium . In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo . Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium . In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion . Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion . Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function . Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels . An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion . However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled . This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo-mediated repression of this transcription. J Bacteriol, 1991 Jul, 173(14), 4247 - 53 Expression of ClpB, an analog of the ATP-dependent protease regulatory subunit in Escherichia coli, is controlled by a heat shock sigma factor (sigma 32); Kitagawa M et al.; Escherichia coli K-12 produces at least two ATP-dependent proteases, Lon (La) and Clp (Ti), the latter consisting of a regulatory subunit (ClpA) and a proteolytic subunit (ClpP) . The gene clpB encoding an analog of ClpA had been found at 57 min on the E . coli chromosome . Cloning and examination of novel heat shock promoters led us to identify a major clpB promoter specifically controlled by a heat shock sigma factor, sigma 32 (the rpoH {= htpR} gene product) . beta-Galactosidase synthesis from a PclpB-lacZ operon fusion was transiently induced upon temperature shift from 30 to 42 degrees C, and the induction depended on the rpoH function . Chromosomal clpB transcripts also increased upon temperature upshift and were totally absent in the rpoH deletion strain . In the in vitro transcription experiments, the clpB promoter was specifically recognized and transcribed by RNA polymerase-sigma 32 . Nucleotide sequencing and determination of mRNA start sites permitted us to identify a major heat shock promoter located upstream of the clpB coding sequence . The results clearly indicate that clpB expression is under direct control of sigma 32 . Since ClpP was recently shown to be a sigma 32-dependent heat shock protein, the present finding suggests the possibility that a potential ATP-dependent protease, ClpB-ClpP complex, plays an important role against thermal stress in E . coli. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5704 - 8 The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev RNA response elements; Bogerd HP et al.; The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for the replication of this pathogenic retrovirus and, surprisingly, can also replace the function of the structurally distinct Rev protein of the type 1 human immunodeficiency virus (HIV-1) . Rex action requires a 255-nucleotide viral RNA stem-loop structure termed the Rex RNA response element (RexRE) located in the 3' retroviral long terminal repeat . Rex function leads to the induced cytoplasmic expression of the incompletely spliced family of viral mRNAs that uniquely encode the HTLV-I structural and enzymatic proteins (Gag, Pol, and Env) . Our studies now demonstrate that Rex acts by binding directly to the RexRE in a sequence-specific manner . These effects of Rex require the presence of a 10-nucleotide subregion of the RexRE that is essential for Rex function in vivo . Dominant-negative mutants of Rex also bind to the RexRE with high affinity, while a recessive-negative Rex mutant altered within its arginine-rich, positively charged domain fails to engage the RexRE . Analogously, both the wild-type and dominant-negative Rex proteins specifically bind to the structurally distinct HIV-1 Rev response element, a finding that likely underlies the respective stimulatory and inhibitory effects of these HTLV-I proteins in the heterologous HIV-1 system . However, consistent with their lack of amino acid homology, the binding sites for Rex and Rev within the HIV-1 Rev response element are distinct. Mol Cell Biol, 1991 Jul, 11(7), 3624 - 32 Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains; Abate C et al.; The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site . Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids . Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro . Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation . Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun . These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex . The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro . In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro . Potentially, the inhibitory domains could serve a regulatory function in vivo . Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains. J Virol, 1991 Jul, 65(7), 3721 - 7 In vitro binding of human T-cell leukemia virus rex proteins to the rex-response element of viral transcripts; Grassmann R et al.; Human T-cell leukemia virus (HTLV-I, HTLV-II) rex protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural proteins . The effect is mediated by a cis-acting rex-response element (RRX) which is located near the 3' end of all viral mRNAs . We show that rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays . Binding analyses with deletion variants of rex proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids . Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion protein containing this peptide bound to the RRX . These results suggest that direct binding of rex protein to the RRX is important for rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of rex to its target RNA. Arch Biochem Biophys, 1991 Jul, 288(1), 225 - 30 Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties; Denisot MA et al.; The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity . Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation . The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label {2-3H}-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function . CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity . These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain. J Dairy Sci, 1991 Jul, 74(7), 2145 - 52 Actions of bovine somatotropin on polymorphonuclear leukocytes and lymphocytes in cattle; Elvinger F et al.; Objectives were to determine 1) in vitro effects of bST on function of polymorphonuclear leukocytes and lymphocytes and 2) in vivo effects of bST on leukocyte function of heifers fed to maintain medium or high growth rates . When administered in vitro, bST did not affect function of polymorphonuclear leukocytes . {Methyl-3H}thymidine incorporation by resting lymphocytes was stimulated by 1000 ng/ml bST . When given in vitro, bST did not further enhance {methyl-3H}thymidine uptake by mitogen-stimulated lymphocytes cultured at 38.5 degrees C but reduced the depression of mitogen-stimulated proliferation caused by incubating cells at 42 degrees C . When bST was administered in vivo, phagocytosis and killing of Escherichia coli by polymorphonuclear leukocytes from bST-treated heifers were not different from cells of control heifers . As measured by {methyl-3H}thymidine uptake after stimulation with phytohemagglutinin, lymphocytes from bST-treated heifers responded similarly to those of control heifers when incubated at 38.5 degrees C, but the depression in {methyl-3H}thymidine uptake due to culture at 42 degrees C was less for lymphocytes obtained from bST-treated heifers . In conclusion, bST had little effect on function of polymorphonuclear leukocytes but could promote proliferation of lymphocytes in vitro and protect cells from effects of elevated temperature. Br Poult Sci, 1991 Jul, 32(3), 589 - 96 Pharmacokinetics of chloramphenicol in normal and Escherichia coli infected chickens; Atef M et al.; 1 . Disposition kinetics were compared in healthy chickens and in chickens naturally infected with E . coli following the intravenous, intramuscular and oral administration of chloramphenicol in a single dose of 20 mg/kg body weight . 2 . Lower serum chloramphenicol concentration in diseased chickens were reported after intravenous injection, but they were higher than normal 30 min after intramuscular and oral administration . Following intravenous injection the volume of distribution was increased in diseased chickens . 3 . The biological half-life in normal chickens was 8.32 +/- 0.5 h and was prolonged in diseased birds (26.21 +/- 0.2 h) . The body clearance of chloramphenicol was reduced in diseased chickens . 4 . The rate of absorption of chloramphenicol was delayed after administration via the oral route but the extent of absorption was increased . The maximum concentration was higher and it was reached after a longer time in diseased than in normal chickens after administration by both intramuscular and oral routes. Appl Environ Microbiol, 1991 Jul, 57(7), 1914 - 9 Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction; Wernars K et al.; The polymerase chain reaction (PCR) was used as a tool for the detection of enterotoxigenic Escherichia coli in minced meat . With two synthetic 29-mer oligonucleotides, a 195-bp fragment from the E . coli heat-labile enterotoxin (LT) gene could be amplified specifically . When 6 CFU was added to the reaction mixture as a template, the PCR yielded sufficient amplified product for visualization on an agarose gel . Prior to PCR amplification, the minced meat samples were subjected to enrichment culturing for E . coli . From these cultures, 10 microliters was used in the PCR assay . All 20 25-g samples that were examined in this assay were negative for E . coli LT . However, when 3 CFU of E . coli LT was added to the 25-g samples of minced meat prior to enrichment culturing, the PCR assay yielded positive results. Am J Vet Res, 1991 Jul, 52(7), 990 - 8 Respiratory, renal, hematologic, and serum biochemical effects of hypertonic saline solution in endotoxemic calves; Constable PD et al.; The respiratory, renal, hematologic, and serum biochemical effects of hypertonic saline solution (HSS) treatment were examined in 12 endotoxic, pentobarbital-anesthetized calves (8 to 20 days old) . Escherichia coli endotoxin (055:B5) was infused IV at a rate of 0.1 microgram/kg of body weight over 30 minutes . Endotoxin induced severe respiratory effects, with marked hypoxemia and increases in arterial-alveolar O2 gradient (P{A-a}O2), physiologic shunt fraction (Qs/Qt), and physiologic dead space to tidal volume ratio (Vd/Vt) . Oxygen consumption was decreased, despite an increase in the systemic O2 extraction ratio . Peak effects were observed at the end of endotoxin infusion . The renal response to endotoxemia was characterized by a decrease in free-water reabsorption and osmotic clearance, as well as a decrease in sodium and phosphorus excretion . Endotoxemia induced leukopenia, thrombocytopenia, hyperphosphatemia, hypoglycemia, acidemia, and increased serum alkaline phosphatase concentrations . Calves were treated with HSS (2,400 mosm/L of NaCl, 4 ml/kg, n = 4) or an equivalent sodium load of isotonic saline solution (ISS; 300 mosm/L of NaCl, 32 ml/kg, n = 4 90 minutes after the end of endotoxin administration . Both solutions were infused over a 4- to 6-minute period . A control group (n = 4) was not treated . Infusion of HSS or ISS failed to induce a significant change in Pao2, P(A-a)O2, (Qs/Qt), (Vd/Vt), or oxygen consumption . Both solutions increased systemic oxygen delivery to above pre-endotoxin values . Hypertonic saline infusion induced significant (P less than 0.05) increases in serum Na and Cl concentrations and osmolality, whereas ISS induced a significant increase in serum Cl concentration and a significant decrease in serum phosphorus concentration.(ABSTRACT TRUNCATED AT 250 WORDS) Anal Biochem, 1991 Jul, 196(1), 19 - 23 Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity; Fitzgerald ML et al.; A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein . A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment . The 32P-labeled nucleotide was located at the penultimate position . The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts . The structure of the released product was characterized on 23% sequencing gels . Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus. Anal Biochem, 1991 Jul, 196(1), 126 - 36 Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo; Frenkel K et al.; Oxidative modification of genetic material has been implicated as a factor in carcinogenesis, particularly during promotion and progression, and therefore there is a need for sensitive detection of oxidized DNA bases . We developed a method that can be applied to DNA isolated from any source and used to simultaneously quantify oxidized nucleosides without a need to prelabel the DNA or use destructive hydrolytic procedures . This method is based on: (a) enzymatic DNA digestion; (b) HPLC separation of the resultant nucleosides; (c) acetylation of the oxidized nucleosides with {3H}Ac2O (acetic anhydride); (d) removal of the radioactive debris; and (e) quantitative analysis of tritiated nucleoside acetates by HPLC . Enzymatic DNA digestion was optimized using DNase I in the presence of Mg2+ (pH 7), followed by nuclease P1 in the presence of Zn2+ (pH 5.1) and alkaline phosphatase (pH 7.5) . Analysis of DNA oxidized with H2O2 in the presence of Fe2+/EDTA for 30 min showed that the levels of 8-OHdG (8-hydroxy-2'-deoxyguanosine) were increased 2.7-fold, HMdU (5-hydroxymethyl-2'-deoxyuridine) 3.15-fold, and FdU (5-formyl-2'-deoxyuridine) 2.5-fold . Although the (-)-isomer of cis-dTG (cis-thymidine glycol) was enhanced 2.3 times, the (+)-isomer remained virtually unchanged . Analysis of DNA isolated from epidermal cells of mice treated in vivo with the tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) showed 4.8-, 2.7-, and 8.7-fold increases in the levels of total cis-dTG, 8-OHdG, and HMdU, respectively, and of some unknown DNA oxidation products . These results prove applicability of the 3H-postlabeling method to the analysis of DNA (and potentially RNA) isolated from many sources, including animals and humans. Somat Cell Mol Genet, 1991 Jul, 17(4), 399 - 410 Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells; Kresnak MT et al.; The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of the Escherichia coli gpt gene integrated into the chromosomal DNA as part of a shuttle vector . Four independently derived GPT- mutants with single base changes within the integrated gpt gene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5' and 3' guanine residues of a GG doublet, the 3' guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet . Two of the mutant lines possessed GG doublet sequences in which a GC----AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence . Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of the gpt genes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC----AT transitions at the 3' guanine residue of the doublet . BrdU-induced reversion of two additional GPT- mutant lines demonstrated that the 3' guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3' guanine residue of a GG doublet or GGGG quartet . All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate . The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene. Circ Shock, 1991 Jul, 34(3), 344 - 8 Influence of pentoxifylline and related analogues in endotoxemic renal failure; Berens KL et al.; Acute kidney dysfunction, manifested by reductions in renal blood flow and glomerular filtration rate with increased renal vascular resistance, is a common finding in septic shock . In an attempt to halt the progressive renal dysfunction, the hemorheologic methylxanthines, pentoxifylline (1, 5, or 10 mg/kg of PTX) and 2 structurally-related analogues, 5 mg/kg of HWA-138 and 5 mg/kg of HWA-448, or saline wee given 7.5 hr after endotoxin infusion in the rat . Renal function, assessed by single-dose inulin clearances (CIN), was measured at 6 hr after the infusion of endotoxin and also 1 hr following the drug treatment . The mean CIN at 6 and 9 hr after endotoxin infusion were 2- and 3-fold decreased compared with control rats given either saline or 5 mg/kg of PTX . Although renal function declined in all rats throughout the study period, the reduction in renal function was markedly slowed in endotoxemic rats given 10 mg/kg of PTX or 5 mg/kg of HWA-448 compared with untreated controls (74 +/- 9% and 77 +/- 9 vs . 47 +/- 12% of 6 hr CIN at 9 hr, respectively; P less than 0.01) . Similar results were found with single doses of 5 mg/kg of PTX or HWA-138; PTX 1 mg/kg had a modest beneficial effect on renal function . There was no evidence of vascular congestion in endotoxemic kidneys upon histologic examination . These data suggest the potential benefit of PTX and related methylxanthines in stopping progressive renal damage associated with septic shock. Biophys J, 1991 Jul, 60(1), 172 - 8 Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle; Lin GC et al.; The rates are determined for the deprotonation and reprotonation of the protonated Schiff base (PSB) as well as of formation and decay of the UV transient in the photocycle of seven bacteriorhodopsin (bR) mutants in which Arg-7, 82, 164, 175, 225, or 227 are replaced by glutamine and Arg-134 by cysteine . The results show that all these mutations increase the rate of deprotonation of the PSB compared to ebR, (wild-type bacteriorhodopsin expressed in Escherichia coli) greatly increase the rate of the reprotonation of the SB (Schiff base) in the case of the Arg-164 and Arg-175 mutations and dramatically decrease this rate in the case of the Arg-227 mutation . Temperature studies on the latter mutant suggest that the observed change in its rate of reprotonation is due to large decrease in the energy and entropy of activation, similar to those observed for Asp-96 mutations (Miller, A . and D . Orsterhelt . 1990 . Biochim . Biophys . Acta . 1020:57-64) . These results suggest that the reprotonation process is changed to a proton diffusion-controlled mechanism in the Arg-227 mutant due to a change in the structure of the proton channel . The absorption intensity ratio (AUV/AMslow) of each arginine mutant relative to that of ebR is found to be similar to that for native purple membrane (PM) except for the Arg-227 mutant where it is greatly reduced, and for the Arg-82 mutant where it is not observed, suggesting that both Arg-227 and Arg-82 residues somehow play roles in inducing the UV transient absorption . All the above results are discussed in terms of the model for the structure of bR proposed by Henderson, R., J.M . Baldwin, T.A . Ceska, F . Zemlin, E . Beckmann, and K.H . Downing . (1990 . J . Mol . Biol . 213:899-929). Plant Mol Biol, 1991 Jul, 17(1), 83 - 8 Production in Escherichia coli of active Sorghum phosphoenolpyruvate carboxylase which can be phosphorylated; Cretin C et al.; Phosphoeolpyruvate carboxylase (PEPC)-deficient mutants of Escherichia coli have been complemented with a plasmid bearing a full-length cDNA encoding the C4-type form of Sorghum leaf PEPC . Transformed cells grew on minimal medium . Two clones were selected which produce a functional and full-sized enzyme protein as determined by activity assays, immunochemical behavior and SDS-PAGE . In addition, regulatory phosphorylation of immunopurified recombinant PEPC was observed when the enzyme was incubated with a partially purified plant PEPC kinase . These results establish that E . coli cells produce a genuine, phosphate-free, higher-plant PEPC . Application of immunoadsorbtion chromatography to bacterial extracts makes it possible to prepare highly pure protein available for biochemical studies. Mol Gen Genet, 1991 Jul, 227(3), 411 - 6 Analysis of barley nitrate reductase cDNA and genomic clones; Schnorr KM et al.; Barley nitrate reductase cDNA and genomic clones were isolated by homology with the barley nitrate reductase cDNA clone bNRp10 and sequenced . This is the first reported analysis of a full-length nitrate reductase gene and its corresponding cDNA in the same species . The longest cDNA clone extends to within 9 bp of the ATG start codon and the sequence is similar to that reported for the higher plant NR sequences . As expected, the amino acid sequence of barley nitrate reductase is more related closely to the rice (84% homology) than to the Arabidopsis (62%) sequence . Four different polyA addition sites were identified from sequence analysis of nine barley NR cDNA clones . A 7.3 kb region of a genomic recombinant lambda clone was subcloned as two contiguous BamHI fragments into p Bluescript, designated pMJ7 and pMJ8, and sequenced . These clones include the entire nitrate reductase coding region, one large intron, 2.7 kb of untranslated sequence 5' to the translation start codon and 0.25 kb 3' to the translation termination codon . The mRNA cap site was identified as a cytosine, 111 bases upstream of the ATG translation start codon . The putative CAAT and TATA boxes were identified at -115 and -33 bp, respectively, with the mRNA cap site designated as +1 . The barley nitrate reductase gene coding region strongly favors G or C in the third codon position. J Cardiovasc Surg (Torino), 1991 Jul-Aug, 32(4), 477 - 81 Recurrent aortic graft infection following descending thoracic aorta to femoral artery bypass . A case report and review; Costantino MJ; This paper presents a patient who developed a recurrent aortic graft infection after a descending thoracic aorta to femoral artery bypass . The patient had previously undergone successful management of an infected aortobifemoral bypass by removal of the graft and revascularization of the lower extremities with axillofemoral bypasses . A general discussion of the management of infected aortic grafts is presented and a discussion of the management of this particular patient is presented in detail. J Gen Virol, 1991 Jul, 72 ( Pt 7), 1543 - 50 Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles; Jagadish MN et al.; When the full-length coat protein (CP) of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed in Escherichia coli or yeast, it assembled to form potyvirus-like particles . The particles were heterogeneous in length with a stacked-ring appearance and resembled JGMV particles in their flexuous morphology and width . This cell-free assembly system should permit analysis of the mechanisms of particle assembly and genome encapsidation . Two mutant forms of CP produced by site-directed mutagenesis failed to assemble into virus-like particles. Biochem J, 1991 Jul 1, 277 ( Pt 1), 263 - 71 D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L . B3728 . Gene cloning, sequence and expression; Loviny-Anderton T et al.; Arthrobacter strain N.R.R.L . B3728 superproduces a D-xylose isomerase that is also a useful industrial D-glucose isomerase . The gene (xylA) that encodes it has been cloned by complementing a xylA mutant of the ancestral strain, with the use of a shuttle vector . The 5' region shows strong sequence similarity to Escherichia coli consensus promoters and ribosome-binding sequences and allows high levels of expression in E . coli . The coding sequence shows similarity to those for other D-xylose isomerases and is followed by 22 nucleotide residues with stop codons in each reading frame, a good 'consensus' ribosome-binding site and an open reading frame showing similarity to those of known D-xylulokinases (xylB) . Studies on the expression of the cloned gene in Arthrobacter and in E . coli suggest that the two genes are part of a xyl operon regulated by a repressor that is defective in strain B3728 . Codon usage in these two genes, and in another open reading frame (nxi) that was adventitiously isolated during early cloning attempts, shows some characteristic omissions and a strong G + C preference in redundant positions. Biochem J, 1991 Jul 1, 277 ( Pt 1), 159 - 63 Expression and site-directed mutagenesis of hepatic glucokinase; Lange AJ et al.; Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli . Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data . The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP . The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family. Biochem J, 1991 Jul 1, 277 ( Pt 1), 153 - 8 Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli; Packman LC et al.; The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques . (1) After the complex had been treated with N-ethyl{2,3-14C}maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl{2,3-14C}maleimide . All the chemically reactive lipoyl groups in the native complex were found to be catalytically active . (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue . Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site . (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate . All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group . (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected . The same result was obtained for the lipoyl domain from the E . coli 2-oxoglutarate dehydrogenase complex . (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error. Am J Pathol, 1991 Jul, 139(1), 199 - 206 Inducible release of an endothelial cell-specific protein; Carson CW et al.; The authors investigated the release of an endothelial cell-specific protein (E92) by cultured porcine aortic endothelial cell cultures . Under normal culture conditions, endothelial cells released little or no E92 into the culture supernatant . Treatment with thrombin (0.01 to 10 units/ml), endotoxin (0.01 to 10 micrograms/ml), or interleukin-1 (0.01 to 3.0 units/ml), however, caused significant, dose-dependent increases in E92 detectable in the culture supernatants . Time-course experiments showed that maximum release of E92 into cellular supernatants occurred 24 hours after stimulation with all mediators . Parallel experiments used 51Cr-loaded endothelial cells as a measure of lethal cellular injury . None of the mediators caused significant injury at the doses observed to induce release of E92 . These results suggest that the release of E92 into the supernatants of cultured endothelial cells is an inducible event . The data also support the hypothesis that detection of E92 antigen in sera from patients with rheumatic disease represents a marker of in vivo vascular endothelial cell activity. Plant J, 1991 Jul, 1(1), 15 - 26 Organ-dependent regulation of a plant promoter isolated from rice by 'promoter-trapping' in tobacco; Claes B et al.; A vector containing a transcriptionally inactive neomycin phosphotransferase II gene was used to select promoter sequences from a pool of random genomic DNA fragments . This paper describes how one such sequence (P4.7) isolated from Oryza sativa acts as a hormonally regulated promoter in Nicotiana tabacum . Relative expression ratios in leaf, root, midrib, callus, and stem tissue of tobacco plants are 1:5:4:10:17 . Histochemical assays show that P4.7 activates the uidA reporter gene throughout the phloem and cortex of tobacco stems . Transcription from the P4.7 fragment is inducible in leaf tissue by low levels of alpha-naphthalene acetic acid or 6-benzyl-aminopurine, even when cell proliferation is inhibited by colchicine or hydroxyurea . Conversely, 1% DMSO was found to inhibit activation of P4.7 without interfering with callus formation . The fragment contains TATA and CAAT sequences normally found at the 5' end of many plant genes, and an additional region homologous to sequences located in similar positions in a variety of similarly regulated promoters . Promoter deletion and fusion experiments have indicated the location of a stem enhancer element in P4.7 . The promoter trap system we have described may potentially be used to characterize transcriptional factors common to monocot and dicot species. Plant Cell, 1991 Jul, 3(7), 719 - 35 Plant enolase: gene structure, expression, and evolution; Van der Straeten D et al.; Enolase genes were cloned from tomato and Arabidopsis . Comparison of their primary structures with other enolases revealed a remarkable degree of conservation, except for the presence of an insertion of 5 amino acids unique to plant enolases . Expression of the enolase genes was studied under various conditions . Under normal growth conditions, steady-state messenger and enzyme activity levels were significantly higher in roots than in green tissue . Large inductions of mRNA, accompanied by a moderate increase in enzyme activity, were obtained by an artificial ripening treatment in tomato fruits . However, there was little effect of anaerobiosis on the abundance of enolase messenger . In heat shock conditions, no induction of enolase mRNA was observed . We also present evidence that, at least in Arabidopsis, the hypothesis that there exists a complete set of glycolytic enzymes in the chloroplast is not valid, and we propose instead the occurrence of a substrate shuttle in Arabidopsis chloroplasts for termination of the glycolytic cycle. Arch Biochem Biophys, 1991 Jul, 288(1), 141 - 4 Site-specific mutagenesis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding; Tait JF et al.; Annexin V (placental anticoagulant protein I) binds tightly to anionic phospholipid vesicles in the presence of calcium . Four mutant proteins were expressed in Escherichia coli in which Ala replaced one of the following residues in the third repeat of annexin V: Arg-200, His-204, Arg-206, or Lys-207 . In a competitive fluorescence quenching assay, the wild-type recombinant protein had the same affinity for phosphatidylserine-containing vesicles as the placentally derived protein . The affinity of the four mutant proteins for phosphatidylserine-containing vesicles was unchanged relative to wild-type protein . We conclude that His-204 and adjacent basic residues, including the highly conserved Arg-200 residue, are not required for high-affinity phospholipid binding. Arch Biochem Biophys, 1991 Jul, 288(1), 107 - 11 Trinitrophenyl-ATP binding to the ArsA protein: the catalytic subunit of an anion pump; Karkaria CE et al.; The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli . Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins . Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with {alpha-32P}ATP . From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein . Purified ArsA protein bound a fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine- 5'-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA . Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein. Mol Gen Genet, 1991 Jul, 227(3), 488 - 92 Properties of RecA441 protein reveal a possible role for RecF and SSB proteins in Escherichia coli; Dri AM et al.; We examined the possibility that the recA441 mutation, which partially suppresses the UV sensitivity of uvr recF mutant bacteria, exerts its effect by coding for an altered RecA protein that competes more efficiently than the RecA+ protein with SSB for ssDNA in vivo . Using an assay measuring recombination between UV-damaged lambda DNA and intact homologous DNA, we found that the introduction of the recA441 mutation partially suppressed the defects in recombination in bacteria lacking RecF activity but not in bacteria with excess SSB, although recombination was affected more in recF mutants than in bacteria overproducing SSB . These results therefore do not support the hypothesis that RecA441 protein, or RecA protein with the help of RecF protein, is required during recombination of UV-damaged DNA to compete with SSB for ssDNA. Mutat Res, 1991 Jul, 249(1), 189 - 93 The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA; Dianov GL et al.; The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay . Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events . DMS treatment also stimulated RecA protein binding to double-stranded DNA . In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination . The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity . The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA . We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA. J Bacteriol, 1991 Jul, 173(14), 4544 - 8 Temperature-sensitive mutations at the carboxy terminus of the alpha subunit of the Escherichia coli F1F0 ATP synthase; Vik SB et al.; Mutations were constructed in the a subunit of the F1F0 ATP synthase from Escherichia coli . Truncated forms of this subunit showed a temperature sensitivity phenotype . We conclude that the carboxy terminus of the a subunit is not involved directly with proton translocation but that it has an important structural role. FASEB J, 1991 Jul, 5(10), 2349 - 60 Molecular biology of the human immunodeficiency virus type 1; Haseltine WA; The immunodeficiency virus type 1 is a complex retrovirus . In addition to genes that specify the proteins of the virus particle and the replicative enzymes common to all retroviruses, HIV-1 specifies at least six additional proteins that regulate the virus life cycle . Two of these regulatory genes, tat and rev, specify proteins essential for replication . These proteins bind to specific sequences of newly synthesized virus RNA and profoundly affect virus protein expression . Tat and rev appear to be prototypes of novel eukaryotic regulatory proteins . These two genes may play a central role in regulating the rate of virus replication . Three other viral genes, vif, vpu, and vpr, affect the assembly and replication capacity of newly made virus particles . These genes may play a critical role in spread of the virus from tissue to tissue and from person to person . Our understanding of the contribution of each of the virus structural proteins and regulatory genes to the complex life cycle of the virus in natural infections is incomplete . However, enough insight has been gained into the structure and function of each of these components to provide a firm basis for rational antiviral drug development. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5597 - 601 Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli; Barnes HJ et al.; When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E . coli membranes . The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E . coli membrane fractions containing the recombinant P45017 alpha enzyme . Surprisingly, it is found that E . coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha . Thus, not only can E . coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo . These studies establish E . coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. J Bacteriol, 1991 Jul, 173(13), 4088 - 94 Biosynthesis of the Escherichia coli K5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product; Finke A et al.; Biosynthesis of the capsular K5 polysaccharide of Escherichia coli, which has the structure 4)-beta GlcA-1,4-alpha GlcNAc-(1, was studied with membrane preparations from an E . coli K5 wild-type strain and from a recombinant K-12 strain expressing the K5 capsule . Polymerization occurs at the inner face of the cytoplasmic membrane without the participation of lipid-linked oligosaccharides . The serological K5 specificity of the in vitro product was determined with a K5-specific monoclonal antibody in an antigen-binding assay . The K5 polysaccharide, as obtained from the membranes after an in vitro incubation, has 2-keto-3-deoxyoctulosonic acid as the reducing sugar, which indicates that the polysaccharide grows by chain elongation at the nonreducing end. J Bacteriol, 1991 Jul, 173(13), 4032 - 8 Multiple effects of Fis on integration and the control of lysogeny in phage lambda; Ball CA et al.; Fis is a small, basic, site-specific DNA-binding protein present in Escherichia coli . A Fis-binding site (F) has been previously identified in the attP recombination site of phage lambda (J . F . Thompson, L . Moitoso de Vargas, C . Koch, R . Kahmann, and A . Landy, Cell 50:901-908, 1987) . The present study demonstrates that in the absence of the phage-encoded Xis protein, the binding of Fis to F can stimulate integrative recombination and therefore increase the frequency of lambda lysogeny in vivo . Additionally, Fis exerts a stimulatory effect on both integration and lysogeny that is independent of binding to the attP F site . Maintenance of the lysogenic state also appears to be enhanced in the presence of Fis, as shown by the increased sensitivity of lambda prophages encoding temperature-sensitive repressors to partial thermoinduction in a fis mutant . In the presence of Xis, however, Fis binding to F interferes with integration by stimulating excision, the competing back-reaction . Since Fis stimulates both excision and integration, depending on the presence or absence of Xis, respectively, we conclude that Xis binding to X1 is the key determinant directing the formation of an excisive complex. J Bacteriol, 1991 Jul, 173(13), 4027 - 31 Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein; Ball CA et al.; The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J . F . Thompson, L . Moitoso de Vargas, C . Koch, R . Kahmann, and A . Landy, Cell 50:901-908, 1987) . We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo . Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells . The defect observed in phage yield is not due to inefficient phage replication or lytic growth . Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis . The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site. J Mol Recognit, 1991 Jul-Dec, 4(4), 129 - 32 Identity elements of Escherichia coli tRNA(Ala); Tamura K et al.; Studies using the T7 transcription system revealed that the discriminator base A73 and the G20 in the variable pocket play important roles in the Escherichia coli alanine tRNA identity . The C60 in the T-loop, which is unique to alanine tRNA, was not found to be crucial for alanine identity . Anticodon replacement into the valine anticodon UAC did not decrease alanine charging activity, and no alanine charging activity was detected in the mutant valine tRNA possessing the alanine anticodon UGC. Mol Biol (Mosk), 1991 Jul-Aug, 25(4), 974 - 88 {Secretion of periplasmic PhoA protein during its supersynthesis into the medium using outer membrane vesicles . Features of chemical composition of the vesicles and membranes of Escherichia coli secreting cells}; Nesmeianova MA et al.; E . coli K12802 cells transformed by multicopy plasmid with phoA gene acquire the ability to oversynthesize alkaline phosphatase, secrete it into the cultural medium, and accumulate the precursor of this enzyme . The dynamics of enzyme production and secretion as well as cytomorphological changes revealed the existence of a mechanism of selective enzyme secretion into the medium . It is characterized by a decrease of enzyme specific activity in periplasm and its increase in cultural medium, appearance of numerous local zones of adhesion of cytoplasmic and outer membranes, formation of large extracellular outer membrane vesicles containing PhoA protein on the cell poles, and their release into the medium . We isolated the vesicles and found that they contain PhoA (in dominating quantity), several other periplasmic proteins, and matrix proteins of outer membranes . By their phospholipid and protein composition, they correspond to the fraction of outer membranes which have the largest density and sedimentation rate and, apparently, contain no lipoprotein. Mol Biol (Mosk), 1991 Jul-Aug, 25(4), 955 - 9 {Mechanism of NADH-sensitized formation of DNA breaks during irradiation with near UV light}; Burchuladze TG et al.; NADH-photosensitized in vitro formation of single-stranded breaks in plasmid DNA pBR322 depends on both the concentration of the sensitizer and the influence of near-UV radiation (320-400 nm) . Scavengers and inhibitors of different activated oxygen species (sodium azide, sodium benzoate, catalase and superoxide dismutase) prevent the formation of breaks in full or partly . The data obtained show that hydroxyl radical (.OH) and singlet oxygen (1O2) are directly involved in the induction of breaks . In this process hydrogen peroxide (H2O2) plays the role of an intermediate in the reaction of .OH formation from superoxide anion-radical (O2-.) which is the first NAD.H-photogenerated product. Microb Pathog, 1991 Jul, 11(1), 1 - 9 Importance of arginine at position 170 of the A subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia coli for toxin activity; Yamasaki S et al.; Comparison of the primary structures of the A subunits of Vero toxin 1 (VT1), Vero toxin 2 (VT2), and two variants of VT2 (VT2vp and VT2vh) and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1) . All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis . To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis . A total of 22 mutants were prepared to examine 14 conserved residues, and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1 . Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically . These results suggest that, in addition to the glutamic acid at position 167 reported previously, arginine at position 170 also plays an important role in the toxin activity of VT1 . A possible chemical mechanism of the enzymatic (N-glycosidase) activity of VT1 is proposed based on the relative activities of various mutants. Arch Esp Urol, 1991 Jul-Aug, 44(6), 746 - 9 {Primary abscess of the psoas muscle}; Ruiz Rubio JL et al.; An additional case of primary psoas abscess is described . The literature is reviewed and its etiopathogenesis, clinical features, diagnosis and treatment are discussed. Actas Urol Esp, 1991 Jul-Aug, 15(4), 393 - 6 {Abscess of the seminal vesicle . Clinico-therapeutic review apropos of a case}; Vallejo Herrador J et al.; Reporting the case of a patient diagnosed with an abscess of the seminal vesicle, treated successfully through parasacral transgluteal percutaneous aspiration lead by computerized axial tomography (CAT) . This represents the first case in the literature of percutaneous access to the seminal vesicles through this route . CAT is an effective diagnostic test which makes non-surgical treatment for this type of abscess simpler . A review is made of the literature with regard to the etiology, diagnosis and therapy of seminal vesicle abscesses. Virus Genes, 1991 Jul, 5(3), 267 - 71 Nucleotide sequence of cDNA encoding the coat protein of beet yellows virus; Brunstedt J et al.; A cDNA clone of beet yellows viral RNA expressed the viral coat protein gene in E . coli . The sequence of the 2724 nucleotide insert revealed three open reading frames, the 3' of which was shown to be the coat protein cistron . This cistron is expressed in E . coli, in spite of there being no obvious ribosome binding site upstream. New Biol, 1991 Jul, 3(7), 709 - 15 Selective elimination of recombinant genes in vivo with a suicide retroviral vector; Plautz G et al.; The ability to express recombinant genes in vivo offers potential new treatments for human disease if questions of safety and toxicity can be addressed . Complications of gene transfer could include, for example, overexpression of introduced genes for growth or angiogenic factors or insertional mutagenesis, both of which could cause uncontrolled cell growth . We report the development of a suicide retroviral vector that provides a method to eliminate cells undergoing rapid growth in vivo . A murine amphotropic retroviral vector was constructed in which the gene for herpesvirus thymidine kinase was included to render proliferating cells sensitive to ganciclovir, and the Escherichia coli beta-galactosidase gene served as a reporter . This vector's efficacy was first assessed in vitro, and beta-galactosidase activity was abolished in several cell lines after treatment with ganciclovir . In vivo, a transplantable murine CT26 adenocarcinoma whose cells were transduced with this vector regressed completely after administration of ganciclovir . In contrast, expression in nondividing cells within rabbit arteries transduced by retroviral infection in vivo was unaffected . This suicide vector therefore eliminates transformed cells but allows survival of normal nondividing cells that express its specific recombinant genes in vivo, and may thus improve the safety and efficacy of gene transfer into living organisms. Prikl Biokhim Mikrobiol, 1991 Jul-Aug, 27(4), 558 - 64 {Role of the energy status of Escherichia coli in cell membrane phospholipid composition changes during aerobic-anaerobic transitions}; Tkachenko AG et al.; The aerobic to anaerobic transition of E . coli is accompanied with interrelated changes of the adenylate pool, energy charge, respiration rate, as well as the phospholipid content of the cell membranes and the activity of the polyamine synthesizing system . The role of the cellular energy status in the control of the relative content of membrane phospholipids is discussed . The control is based either on energy redistribution in phospholipid metabolism or on the effect on the activity of the polyamine synthesizing system. Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 29 - 32 {Sensitizing properties of Brucella protein antigens synthesized in K12 Escherichia coli cells}; Malikov VE et al.; The sensitizing properties of brucella 31 kD and 31+15 kD protein antigens produced by Escherichia coli cell carrying and expressing the corresponding brucella genes were compared . In experiments evaluating the test of mouse feet oedema in CBA line animals the property of the 31 kD antigen preparation to induce the specific oedema effect was demonstrated on the level comparable with the one produced by the brucella outer membrane proteins preparation . The obtained data were confirmed in the experiments on adoptive transfer of prolonged type hypersensitivity via the spleen cells from the sensitized donors to intact recipient animals . The future of molecular cloning technique usage for obtaining the homogeneous stable preparations of brucella antigens with low reactivity and high specificity is discussed. Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 20 - 2 {Determination of the initial rate of antimutagenic repair in UV-irradiated WP2 Escherichia coli cells}; Filippov VD et al.; The initial rates of antimutagenic dark repair were measured in Escherichia coli WP2 trpE65 cells irradiated by UV-light (11 J/m2) and then incubated in liquid media of various compositions . Samples were taken from suspension of incubated bacteria every 5 min following irradiation, mixed with acriflavine to block further repair and plated onto the selective medium containing acriflavine (1 micrograms/ml) to score the Trp+ mutations . The initial rate of antimutagenic repair was estimated from the kinetics of disappearance of mutations in several successive probes . It appeared to depend on the composition of a medium, to establish just after placing irradiated bacteria onto the medium and to decrease significantly in irradiated cells incubated under conditions favourable for growth . The decrease was not due to inhibition of postreplicative repair and was not caused by casaminoacids as such, but by combination of growth factors that provided the intensive protein synthesis . The decrease could be responsible for a strong mutational response of bacteria to irradiation because it secures the survival of premutagenic lesions in DNA till mutation fixation . It is suggested that metabolic regulation of the antimutagenic repair activity exists, based on an active switch of the energy flows required for several parallel metabolic pathways that proceed in irradiated cells. Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 12 - 5 {Features of the interaction of Escherichia coli and Francisella tularensis RNA polymerases with hybrid plasmids bearing fragments of Francisella tularensis chromosomal DNA}; Pomerantsev AP et al.; Hybrid plasmids containing the fragments of Francisella tularensis chromosomal DNA and capable of tet-gene expression both in Escherichia coli and Francisella tularensis cells were constructed . The regions of francisella chromosomal DNA binding the RNA-polymerases of Escherichia coli and Francisella tularensis were found by the electron microscopy technique . Interconnection of those regions with the expression of tet-gene of the hybrid plasmids was demonstrated. Biochimie, 1991 Jul-Aug, 73(7-8), 947 - 60 The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation; Nagano K et al.; Lim and Spirin {25} proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation . Spirin and Lim {26} excluded the possibilities of the side-by-side model proposed by Johnson et al {13} and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus {3} . However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge . With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al {19} . In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA . The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation . The A76 of A site tRNA overlaps the binding region of puromycin . The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA {14} and 17 A from the 5' end of the anticodon of A site tRNA {15} . It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally {6} . A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model. Biochimie, 1991 Jul-Aug, 73(7-8), 919 - 25 Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy; Tumminia SJ et al.; Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles . Variations in composition, relative molecular mass (Mr) and shape were observed both in the monosomes and in the ribosomal subunits . None of these changes can be resolved visually; they can be evaluated only by computer processing . The variations in relative mass and shape monitored by values of radius of gyration (RG) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity . The highest activity was found in monosomes prepared from the standard 0.5 M NH4Cl wash . With increasing concentrations (up to 1.5 M) of NH4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH4Cl . The most striking effects were observed in ribosomal particles washed with 0.1 M NH4Cl . The 70S monosomes and the 30S subunits attained maximum Mr and RG values (2660 kDa and 76 A, and 990 kDa and 75 A, respectively), which were greater than the theoretical values, while the activity was minimal (approximately 12%) . The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (approximately 1600 kDa and 68 A). Biochimie, 1991 Jul-Aug, 73(7-8), 1121 - 9 A rRNA-mRNA base pairing model for UGA-dependent termination; Prescott CD et al.; A series of site-directed mutations has been constructed in E coli 16S rRNA and shown to suppress UGA-dependent translational termination . With the exception of the C726 to G base change, all were constructed in helix 34 . Characterization of these mutations is reviewed here and from these data and mRNA-rRNA base pairing model for the termination event is presented . The interaction functions via antiparallel base pairing between either 1 of the 2 UCA motifs in helix 34 and the complementary UGA stop codon on the message, thus forming a quasicontinuous A-type helical structure that is further stabilized by stacking enthalpy . Finally, rRNA motifs potentially required for UAA and UAG-dependent translational termination are discussed. Biochimie, 1991 Jul-Aug, 73(7-8), 1113 - 20 Interaction of the release factors with the Escherichia coli ribosome: structurally and functionally-important domains; Moffat JG et al.; There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome . RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site . This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11 . The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive . The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain . Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits . A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain . Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome. Biochimie, 1991 Jul-Aug, 73(7-8), 1051 - 9 Structure-function relationships of elongation factor Tu as studied by mutagenesis; Anborgh PH et al.; We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships . The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule . So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins . In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis . In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate . SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity . Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP) . Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS) Biochimie, 1991 Jul-Aug, 73(7-8), 1045 - 50 How many EF-Tu molecules participate in aminoacyl-tRNA binding? Bensch K, Pieper U, Ott G, Schirmer N, Sprinzl M, Pingoud A. The stoichiometry of the EF-Tu-GTP-aminoacyl-tRNA complex has been re-determined by a variety of methods, viz gel filtrations, fluorescence titrations, as well as hydrolysis and RNase protection experiments . The results of these experiments clearly demonstrate that one aminoacyl-tRNA interacts with only one EF-Tu-GTP molecule, in agreement with the established view and in contrast to the recently published results by Ehrenberg et al {6}. Antonie Van Leeuwenhoek, 1991 Jul, 60(1), 7 - 11 Analysis of the promoter region in the rRNA operon from Mycobacterium bovis BCG; Suzuki Y et al.; At least two transcriptional initiation sites were observed in rRNA operon of Mycobacterium bovis BCG at approximately -187 and -265 . The former transcriptional signal was recognized by Escherichia coli RNA polymerase, whereas the later was not. J Protozool, 1991 Jul-Aug, 38(4), 329 - 34 Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica; Tachibana H et al.; Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS . When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E . histolytica trophozoites . Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites . Two of the remaining five monoclonals reacted strongly with trophozoites of the E . histolytica-like Laredo strain . The determinant antigen was located in the cytoplasm . The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E . histolytica and E . histolytica-like Laredo, E . hartmanni, E . coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis . These three antibodies were also reactive with T . vaginalis and mammalian cells such as HeLa cells . Thus, the combined use of monoclonal antibodies seems capable of distinguishing E . histolytica and/or E . histolytica-like Laredo from other enteric protozoa. Biochimie, 1991 Jul-Aug, 73(7-8), 991 - 1000 Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex; Wakao H et al.; The specific effect of the binding of initiation factor IF2 on E coli 16S rRNA within the {IF2/30S/GTP} complex has been probed by crosslinking experiment with trans-diamminedichloro platinum (II) and by phosphate alkylation with ethylnitrosourea . Several 16S rRNA fragments crosslinked to IF2 have been identified and are mostly located in the head and the lateral protrusion of the 30S subunit . The study of the effect of IF2 binding to the 30S subunit reveals that the factor does not tightly bind to the 16S rRNA and induces both isolated reductions and enhancements of phosphate reactivity in the 16S rRNA . Several of them are located near the binding site of IF2 and weak effects are observed in distant parts of the subunit . These results are discussed in the light of current knowledge of the topographical localization of IF2 with the 30S subunit and of its relation with function. Biochimie, 1991 Jul-Aug, 73(7-8), 961 - 9 A consonant model of the tRNA-ribosome complex during the elongation cycle of translation; Wower J et al.; Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coli ribosome . Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links . As a result, we propose a new model of the tRNA-ribosome complex that accounts for nearly all of the pertinent evidence. Biochimie, 1991 Jul-Aug, 73(7-8), 927 - 36 RNA-protein interactions in the Escherichia coli ribosome; Brimacombe R; Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome . These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA . This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli. Biochimie, 1991 Jul-Aug, 73(7-8), 911 - 8 Localization of a segment of 16S RNA on the surface of the small ribosomal subunit by immune electron microscopy of complementary oligodeoxynucleotides; McWilliams RA et al.; Oligonucleotides that complement Escherichia coli 16S ribosomal RNA residues 685-696 and 694-705 have been synthesized so as to incorporate antibody-recognizable markers: a 3'-terminal residue of N6-delta 2-isopentenyladenosine, a 5'-dinitrophenyl group, or both . Each oligonucleotide is able to bind RNA within the small ribosomal subunit, whether free or in 70S ribosomes . Immune electron microscopy places probes at nucleotides 685, 694 and 705 within a single area, at the tip of the subunit platform, very near the position of the 3'-end of the 16S RNA. Biochimie, 1991 Jul-Aug, 73(7-8), 899 - 910 Frozen spin targets in ribosomal structure research; Stuhrmann HB; Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation . A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction . Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins . Pure deuteron spin labels and proton spin labels are created by NMR saturation . We report on results obtained from the large subunit of E . coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN . The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation . Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K . At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target) . Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively . The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature . This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs. Biochimie, 1991 Jul-Aug, 73(7-8), 1061 - 6 Is there a unique ribosome phenotype for naturally occurring Escherichia coli? Mikkola R, Kurland CG. We have compared the growth characteristics of natural isolates of E coli with the kinetic properties of their ribosomes in vitro . The variability of the different performance characteristics of ribosomes isolated from natural isolates of E coli show that there is not a unique wild-type ribosome phenotype just as there is not a unique growth phenotype for the bacteria . In addition, there is a strong correlation between the growth rates and the efficiency of the kinetic interaction between the ribosomes and the EF-Tu-GTP-aminoacyl-tRNA ternary complex in vitro . No such correlation is seen between the growth rate and the maximum turnover rate of the ribosomes in vitro . These data suggest that the codon-programmed ribosomes are not kinetically saturated with ternary complexes in vivo. Biotechniques, 1991 Jul, 11(1), 40 - 4 Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification; Frothingham R et al.; Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms . We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing . Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification . Sequence was obtained from a crude lysate from a single colony of bacteria . The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications. J Cardiovasc Pharmacol, 1991 Jul, 18(1), 111 - 9 Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model; Martin U et al.; We studied the thrombolytic dose-response relationship of a recombinant plasminogen activator (rPA) (BM 06.022) compared with alteplase in a canine model of coronary artery thrombosis . BM 06.022 consists of the kringle 2 and protease domains of human tissue PA (tPA) and lacks oligosaccharide side chains because of its expression in Escherichia coli . Thrombus formation in anesthetized, open-chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery in the presence of a critical stenosis . Intravenous bolus injection of BM 06.022 (50, 100, 140, and 200 kU/kg) or of alteplase (200, 800, 1,130, and 1,600 kU/kg) 30 min after coronary occlusion to six heparinized dogs per group achieved a dose-dependent increase in reperfusion rate and decrease in residual thrombus wet weight . Vehicle-treated dogs did not reperfuse . Semilogarithmic regression analysis showed that the effective dose that produced 50% reperfusion of BM 06.022 (83 kU/kg) was 11.6-fold lower than that of alteplase (951 kU/kg) . Comparison with infusion experiments showed that intravenous bolus injection of 140 kU/kg of BM 06.022 was equieffective to a 90-min infusion of 800 kU/kg (= 1 mg/kg) of alteplase as a standard treatment regarding reperfusion rate (66%) and time to reperfusion (15 +/- 6 vs . 18 +/- 8 min) . Pharmacokinetic analysis for functionally active BM 06.022 or alteplase in plasma revealed a total plasma clearance of 4.1-6.6 ml/min/kg for BM 06.022 and of 12.6-42.3 ml/min/kg for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS) Vaccine, 1991 Jul, 9(7), 477 - 84 Epitopes of the human malaria parasite P . falciparum carried on the surface of HBsAg particles elicit an immune response against the parasite; von Brunn A et al.; The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function . B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence . Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length . Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals . Monoclonal antibodies derived from such infections recognize the native parasite. J Neurobiol, 1991 Jul, 22(5), 443 - 61 Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development; Robinow S et al.; The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system . Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages . A product of this gene, the ELAV protein, has homology to known RNA binding proteins . The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli . In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia . These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen . We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance. Anal Biochem, 1991 Jul, 196(1), 174 - 7 Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: application in determining epitope accessibility on ribosomes; Syu WJ et al.; A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed . The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined . We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay . Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes . This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes . Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies . Thus, the relationship of different accessible epitopes in situ can be readily examined . Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems. Immunology, 1991 Jul, 73(3), 298 - 303 Biochemical and developmental characterization of the murine cluster of differentiation 1 antigen; Mosser DD et al.; The cluster of differentiation-1 (CD1) antigens are major histocompatibility complex (MHC) class I-like glycoproteins belonging to the immunoglobulin supergene family . Initially described in humans, more recently putative CD1 encoding genes have been identified in several other species, including the mouse where it has been clearly demonstrated that CD1 mRNA is expressed . However, in the mouse both its unusually wide tissue distribution and the prevalence of incompletely spliced RNA have raised the possibility that the mRNA did not encode a functional protein . We have utilized a rabbit polyclonal antiserum raised against an Escherichia coli-expressed recombinant murine CD1 fusion protein to characterize the murine CD1 protein . Here we demonstrate that the antiserum binds specifically to a set of glycoproteins (49,000-55,000 MW) which contain a common core protein with both a size (36,000 MW) and tissue distribution in accordance with those predicted . During thymic ontogeny, this protein is highly expressed by Day 14 of embryonic development and persists into adulthood, while its pattern of expression in other organs changes significantly during development . Thus, the mouse provides an amenable model system for the study of CD1 function. APMIS, 1991 Jul, 99(7), 615 - 9 Complex formation between Escherichia coli lipopolysaccharide O antigen and capsular K antigen as detected by immunoelectrophoresis; Orskov F et al.; Many Escherichia coli of serotypes commonly found in the normal intestine and in extraintestinal diseases, and having capsular antigens of the low molecular group called group II, will, in simple saline extracts, produce complexes between some or all of the lipopolysaccharide molecules and some of the polysaccharide K molecules . Non-complex-forming and complex-forming strains with the same O and K can be found . The complexes are thermostable but are disrupted by some detergents . O-K complex formation may lead to misinterpretation of immunoprecipitation results; one example is the counter current technique used for K determination of E . coli . In this technique O antigen lipopolysaccharide may, when complexed to K polysaccharide, mimic a K antigen . The possible implications of O-K complex formation during the infection process, especially for antibody formation need to be examined. J Bacteriol, 1991 Jul, 173(14), 4297 - 309 Isolation, transcription, and inactivation of the gene for an atypical alkaline phosphatase of Synechococcus sp . strain PCC 7942; Ray JM et al.; The alkaline phosphatase of Synechococcus sp . strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli . The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases . Some sequence similarities were observed between the Synechococcus sp . strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E . coli . Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding . Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus . The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min . Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately . The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein . Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene . Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake. FASEB J, 1991 Jul, 5(10), 2361 - 8 Regulation of HIV-1 gene expression; Cullen BR; The quantity and quality of HIV-1 gene expression is temporally controlled by a cascade of sequential regulatory interactions . Basal HIV-1 transcription is determined by interaction of cellular regulatory proteins with specific DNA target sequences within the HIV-1 long-terminal repeat . The most notable of these protein:DNA interactions involves NF-kappa B, a transcription factor that plays a pivotal role in the activation of genes important for cellular responses to infection and inflammation . A second level of control involves the virally encoded Tat trans-activator . Tat, in combination with as yet unidentified cellular proteins, activates HIV-1 gene expression through a specific interaction with the viral TAR RNA stem-loop target sequence . A final level of regulation is mediated by the viral Rev protein . Rev acts posttranscriptionally to induce the expression of HIV-1 structural proteins and thereby commits HIV-1 to the late, cytopathic phase of the viral replication cycle . Rev activity appears to require a critical, threshold level of Rev protein expression, thus preventing entry into this late phase in cells that are unable to support efficient HIV-1 gene expression . In total, this cascade of regulatory levels allows the HIV-1 provirus to respond appropriately to the intracellular milieu present in each infected cell . In activated cells, the combination of Tat and Rev can stimulate a very high level of viral gene expression and replication . In quiescent or resting cells, in contrast, these same regulatory proteins are predicted to maintain the HIV-1 provirus in a latent or nonproductive state. Vet Med (Praha), 1991 Jul, 36(7), 423 - 31 {Antibody response in mice, rabbits and pigs in response to vaccination with an inactivated oil vaccine containing rotavirus and Escherichia coli strains with K88, K99 and 987P fimbrial antigens}; Zuffa A et al.; In trials with mice, rabbits and weanling piglets, four experimental charges of a combined inactivated oil vaccine against diarrhoeas in mammals were tested: the vaccine was to be implanted to sows and it contained porcine rotavirus (PRV); two charges also contained bovine rotavirus and bacterins of enterotoxicogenic strains of E . coli with protective antigens K88, K99 and 987P . At low starting antibody titres the twofold i.m . implantation of 0.2 ml vaccine stimulated in mice the production of antibodies to reach the average titre value of 1:128 against PRV and of 1:256 against BRV; in rabbits the twofold i.m . implantation of 2 ml vaccine stimulated the antibody development to reach the average titres of 1:508 or 1:500, and in weanlings after the twofold i . m . implantation of the vaccine the titres were 1:1028 or 1:469; in mice agglutination antibodies to antigen K88 had the average value of 1:68, to antigen K99 the value of 1:44 and to antigen 987P the value of 1:8192; in rabbits the respective titres were 1:285, 1:136 and 1:6006 and in pigs 1:570, 1:631 and 1:8192 . The antibodies to antigen 987P persisted at the same level in pigs for six months . Even though there was a gradual decrease in the antibodies to antigens K88 and K99, at that time the values were 9.8 times, or 15.2 times higher than the starting values, and only the antibodies to PRV dropped to the pre-vaccination level . Repeated administration of vaccine to pigs after six months from revaccination induced, with the exception of antigen 987P, an increase in antibodies in a fortnight to reach such titres that were recorded after revaccination. Res Microbiol, 1991 Jul-Aug, 142(6), 653 - 8 Nucleotide sequence of the genes coding for minor fimbrial subunits of the F1C fimbriae of Escherichia coli; van Die I et al.; F1C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces . This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae . The foc gene cluster encoding F1C fimbriae has been cloned, as described previously . Here we present the nucleotide sequence (2081 bp) coding for the F1C minor fimbrial subunits . The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids . The deduced amino acids of the F1C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae . The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower. J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1591 - 601 Adhesion of K99 fimbriated Escherichia coli to pig intestinal epithelium: correlation of adhesive and non-adhesive phenotypes with the sialoglycolipid content; Seignole D et al.; Evidence for the existence of two phenotypes of piglets born to experimental herds was obtained based on the susceptibility of intestinal brush borders to adhesion of K99-positive Escherichia coli . The enterocytes of the K99-receptive piglets displayed a characteristic sialoglycolipid pattern, with a higher content of the monosialoglycolipids II3NeuGc-LacCer (GM3Gc), IV3NeuGc-nLcOse4Cer (SPGGc) and IV3NeuAc-nLcOse4Cer (SPG) and the oligosialogangliosides IV3NeuAc,II3NeuAc-GgOse4Cer (GD1a), II3(NeuAc)2-GgOse3Cer (GD2), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3(NeuAc)2-GgOse4Cer (GT1b) when compared to the gangliosides of non-receptive piglets . The gangliosides from enterocytes of the non-receptive piglets were mainly the monosialogangliosides II3NeuAc-GgOse3Cer (GM2) and II3NeuAc-LacCer (GM3), only traces of the other sialoglycolipids being detected . Adhesion of 14C-labelled K99-positive E . coli cells to the piglet small intestinal sialoglycolipids, as tested by the thin-layer chromatogram overlay assay, revealed that the receptive enterocyte membrane was richer in glycolipids containing K99 receptor structures than the non-receptive enterocyte . Adhesion of K99-positive E . coli correlated with the degree of sialylation of the brush border glycolipids. Zentralbl Veterinarmed B, 1991 Jul, 38(5), 373 - 81 Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases . IV . In vitro studies on protective effects against adhesion of enterotoxogenic Escherichia coli to isolated enterocytes; Jungling A et al.; The pathogenic effect of enterotoxogenic E . coli is mainly to be found in the jejunum . Hence, an effective immunoprophylaxis should be focussed at this site . This can be done effectively by an oral application of specific antibodies . The present study describes the effectiveness of specific antibodies against E . coli . The antibodies were gained from the yolk of E . coli immunized hens and tested for their in vitro activity . The tests showed a significantly reduced adhesion of enterotoxogenic E . coli onto isolated pig enterocytes as long as the bacteria had been incubated with the specific yolk antibodies beforehand . For this reason, yolk immunoglobulins from immunized hens prove to be an effective and handy source of specific antibodies which offers good protection against adhesion of E . coli onto pig enterocytes. J Clin Microbiol, 1991 Jul, 29(7), 1339 - 43 Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis; Tyler SD et al.; A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively . An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes . Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms . Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure . Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b . For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b . Two strains of E . coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes . The VT2 reference strain of E . coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E . coli, pEB1, possessed only that of the VT2 gene . The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype. J Urol, 1991 Jul, 146(1), 217 - 22 Binding sites for P and/or type 1-piliated Escherichia coli in human ureter; Fujita K et al.; Adherence sites for uropathogenic Escherichia coli in the excised human ureter were studied by using scanning electron microscopy . P-piliated E . coli adhered to younger epithelial cells which had microvilli on their surfaces, but did not to mature epithelial cells which had microfolds on their surfaces . This adherence was D-mannose resistant and alpha-D-Galactopyranosyl-(1----4)- beta-D-Galactopyranoside sensitive . Type-1 piliated E . coli adhered to both types of epithelial cells, but it was prevented by D-mannose . Entrapment of adherent type 1-piliated E . coli was observed only on the epithelial cells with microfolds . This model system allowed quantitative estimates of bacterial adherence to the luminal surface of the human ureteral mucosa in vitro, and demonstrated different manners of adherence of P and type 1-piliated E . coli. Infect Immun, 1991 Jul, 59(7), 2485 - 93 Binding of nonspecific cross-reacting antigen, a granulocyte membrane glycoprotein, to Escherichia coli expressing type 1 fimbriae; Sauter SL et al.; Nonspecific cross-reacting antigen (NCA) is a well-characterized membrane glycoprotein on granulocytes, macrophages, and lung epithelium . Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin supergene family, and hybridization studies showed that the transcript level of NCA is induced by treatment with gamma interferon . These studies, as well as the expression of NCA on granulocytes, suggest a role for NCA in immune response . For a first step in studying this possible role, we have examined the binding of two glycoforms of NCA designated NCA-50 (Mr, 50,000) and TEX-75 (Mr, 75,000) . Here we report the results from binding assays which demonstrate carbohydrate-mediated binding of Escherichia coli expressing type 1 fimbriae and of isolated type 1 fimbriae to NCA-50 . TEX-75 did not bind to the purified fimbriae but bound slightly to the bacterial strain . Inhibition studies showed that the binding to NCA-50 involved interaction of mannose moieties on NCA-50 and lectins on the fimbriae . The binding of NCA-50 to bacterial fimbriae was confirmed by electron microscopy studies, using immunolabeling techniques . In addition, we show that the surface expression of NCA-50 (and presumably of other NCA species) on isolated polymorphonuclear leukocytes is increased following activation with the bacterial peptide formylmethionyl-leucyl-phenylalanine, consistent with a role for NCA in immune response. Infect Immun, 1991 Jul, 59(7), 2333 - 40 An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells; Korth MJ et al.; A genetic determinant related to that encoding the F41 fimbrial adhesin was cloned from a bovine septicemic isolate of Escherichia coli . This determinant was found to mediate expression of morphologically distinct fimbriae in E . coli HB101 . The gene encoding the fimbrial subunit protein was identified, and the nucleotide sequence was determined . Homology with the amino-terminal amino acid sequence of CS31A (J . Girardeau, M . Der Vartanian, J . Ollier, and M . Contrepois, Infect . Immun . 56:2180-2188, 1988) was observed, suggesting that this determinant encodes expression of the CS31A fimbrial antigen . The CS31A subunit gene was found to share extensive homology in its signal sequence to the subunit genes encoding the F41 and K88 adhesins . No apparent homology between the mature F41 and CS31A subunits was identified . However, substantial relatedness to the K88 fimbrial subunit was observed . Analysis of the protein products encoded by the CS31A, F41, and K88 determinants in maxicells established that despite extensive genetic similarities between the determinants, each encodes a distinct profile of proteins . E . coli HB101 harboring the cloned CS31A determinant was found to adhere to epithelial cells in a tissue culture assay, suggesting a role for CS31A in adherence . A CS31A-specific DNA hybridization probe detected homologous sequences among enterotoxigenic as well as septicemic E . coli isolates from calves. Biofizika, 1991 Jul-Aug, 36(4), 700 - 2 {Plant alkaloid from Ammopiptantus mongolia--an inhibitor of nitrogen oxide synthesis in animals}; Burgedbazar B et al.; A decrease of the formation in the mouse liver of nitrogen oxide incorporated into ferrum mononitrozyl complexes (FMNC) with diethyldithiocarbamate (DETC) recorded by ESR method was discovered . This decrease was induced by one of the alkaloids isolated from Ammopiptantus mongolica which grows in the Gobi desert . This effect seems to be due to the antioxidant properties of the alkaloid under study . Alkaloid lessened the formation of FMNC with DETC both in the control animals and in those treated with lipopolysaccharide from E . coli initiating inflammation processes and intensification of NO synthesis . Proceeding from the data obtained it is suggested that free radicals reacting with the antioxidant affect NO formation by increasing the level of free calcium in the cell. Virus Genes, 1991 Jul, 5(3), 243 - 54 A second group of hepatitis C viruses; Tsukiyama-Kohara K et al.; cDNA clone 11-7 was isolated by immunoscreening a cDNA library that was prepared from a pooled plasma of non-A non-B hepatitis (NANBH) patients using expression vector lambda gt11 . This cDNA corresponds to known nucleotide positions 3983-4745 of the genome of hepatitis C virus (HCV) . This clone was used as a probe for screening the HCV-related cDNAs in a cDNA library similarly prepared by using lambda gt10 . As a result, six more cDNA clones were isolated and analyzed for their nucleotide sequences . The results strongly suggested that there are at least two groups of HCV, group I and group II . According to our classification, the prototype HCV and clone 11-7 belong to group I HCV, and their nucleotide and deduced amino acid sequences were diverged from those of group II HCV . Genetic variation observed in the nucleotide and the amino acid sequences between the two groups resembles that in the NS3 region of the genome between Japanese encephalitis virus and West Nile fever virus . Polypeptides produced in Escherichia coli carrying a clone 11-7 or a group II cDNA clone E reacted with antibodies in the blood of 12 or 4 out of 14 individual chronic NANBH patients, respectively . Our data clearly indicate the existence of a second group of HCV. Res Microbiol, 1991 Jul-Aug, 142(6), 633 - 41 An Escherichia coli insertion element (IS2) provides a functional promoter in Bordetella pertussis; Goyard S et al.; The adenylate cyclase (cyaA) gene of Bordetella pertussis is not expressed in Escherichia coli . Using cya-lac fusions, high-expression spontaneous mutants were isolated and shown to have the insertion element IS2 in orientation II integrated into the reading frame of cyaA . Upon transfer of the IS2-activated cya-lac fusion into B . pertussis, we found that the IS2-provided promoter is as efficient in B . pertussis as it is in E . coli . These results provide evidence that an insertion element derived from the E . coli chromosome can activate gene expression in B . pertussis, a taxonomically distant organism. Zhonghua Yi Xue Za Zhi, 1991 Jul, 71(7), 373 - 7, 26 {Experimental study of the protective effects of N-acetylcysteine on endotoxin-induced acute lung injury}; Zhu Y; The purpose of this study was to investigate the effects of NAC (N-acetylcysteine) on endotoxin induced acute lung injury in unanesthetized sheep . The results showed that NAC attenuated the responses to endotoxemia, and the rise in pulmonary artery pressure was significantly diminished, and the rise of TXB2 and 6-keto-PGF1 alpha in plasma and lung lymph was less significantly for NAC + E than for E alone . The QL (lung lymph flow), PC (lung lymph protein clearance) and PS (pulmonary capillary surface permeability area) were significantly decreased in NAC + E group . The lipid peroxide contents in artery plasma and lung lymph were not increased in NAC-treated group . We conclude that NAC is capable of attenuating all pathophysiologic changes after endotoxin infusion, blocking the reaction of oxygen free radicals, and protecting lungs from oxidant damage. Plasmid, 1991 Jul, 26(1), 40 - 54 Construction and characterization of derivatives carrying insertion mutations in F plasmid transfer region genes, trbA, artA, traQ, and trbB; Kathir P et al.; We devised a method for construction of insertion mutations in F plasmid tra region genes as a means of investigating the functions associated with previously uncharacterized loci . First, we constructed mutations in vitro, by insertion of a kanamycin resistance gene into a unique restriction site within a tra region fragment carried by a small, chimeric plasmid . Second, we crossed the insertion mutations, in vivo, onto a plasmid containing the complete F tra region sequence (either F lac, or pOX38, a Tra+ F plasmid derivative) . Using this method, we obtained F lac mutant derivatives carrying KmR gene insertions in traQ, and a set of pOX38 mutant derivatives carrying a KmR gene insertion in trbA, artA, traQ, or trbB . Analysis of these derivatives showed that insertion of a kan gene at the NsiI site of traQ resulted in transfer deficiency, F-pilus-specific-phage resistance and an absence of detectable F-pilin subunit synthesis . Since the traQ mutants regained a wild-type phenotype when complemented with a traQ+ plasmid clone, we concluded that traQ expression is essential to transfer and F-pilus synthesis . However, pOX38 derivatives carrying kan gene inserts in genes trbA, artA, or trbB retained F-pilus-specific phage sensitivity and transferred at normal levels . Thus, these three gene products may not be essential for F-transfer from Escherichia coli K-12 under standard mating conditions. Cell Biol Int Rep, 1991 Jul, 15(7), 595 - 606 An optimum dose of c-H-ras is a prerequisite for hormone-dependent conversion of a cell between cancerous and normal states in tissue culture; Xu ZZ et al.; We have established a few cell lines which can be reversibly converted from cancerous to normal and vice versa by the addition to, or removal from the culture medium of glucocorticoid hormone . These cell lines were derived from mouse NIH 3T3 cells and possessed the integrated gene pairs on chromosomes, which are composed of human mutated c-H-ras fused with mouse mammary tumour virus long terminal repeat and E . coli xanthine-guanine phosphoribosyltransferase gene with the SV40 promoter . We have characterised these cell lines in order to elucidate an essential requirement for the conversion of the state of a cell . It was found that the presence of at least two to three copies of the gene pair per diploid genome are essential . An approximate threshold level of c-H-ras 1.6 kb RNA required for reversible conversion was estimated. Acta Physiol Scand, 1991 Jul, 142(3), 375 - 86 Segmental heterogeneity of the rat colon in the response to activators of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway; Nobles M et al.; The electrolyte transport was compared in proximal and distal segments of the rat colon under control conditions and after induction of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway . Baseline short-circuit current was decreased by indomethacin and tetrodotoxin in the distal colon, indicating a spontaneous production of neuronally acting prostaglandins . In contrast, baseline short-circuit current in the proximal colon was decreased only by indomethacin, but not by tetrodotoxin . Unidirectional flux measurements revealed that in the distal colon sodium and chloride were absorbed, while the proximal colon secreted chloride . A morphological comparison between the distal and proximal epithelium revealed that the zonulae occludentes and the microvilli were longer in the distal colon . The size of the Golgi apparatus was several times larger in the crypt than in the surface region without differences between proximal and distal colon . Distal segments were more sensitive to an activator of the Ca(2+)-pathway, carbachol, or activators of the cAMP-pathway such as forskolin and a cAMP-analogue . In contrast, the activation of the cGMP-pathway by a cGMP-analogue or by the heat-stable enterotoxin of E . coli (STa) was more effective in the proximal colon . The results give evidence for a segmental specificity with regard to the intracellular pathways responsible for the activation of secretion. J Appl Physiol, 1991 Jul, 71(1), 342 - 51 Pentoxifylline does not attenuate acute lung injury in the absence of granulocytes; Yonemaru M et al.; Pentoxifylline (PTX), a methylxanthine, can suppress polymorphonuclear leukocyte (PMN) activation and attenuate sepsis-induced acute lung injury . We investigated whether PTX prevents non-PMN-dependent lung injury . First we studied four groups of granulocyte-depleted guinea pigs (control, PTX, Escherichia coli, and E . coli + PTX) . Lung injury was assessed by wet-to-dry lung weight (W/D) ratio and lung tissue-to-plasma 125I-albumin ratio (albumin index, AI) . The E . coli group showed a significant increase in the lung W/D ratio and AI compared with the control and PTX groups . However, PTX did not prevent the E . coli-induced increase in the lung W/D ratio and AI . Next we investigated the effects of PTX on endothelial cell monolayer permeability and adenosine 3',5'-cyclic monophosphate (cAMP) levels . Whereas E . coli lipopolysaccharide (LPS) alone increased the endothelial permeability, PMNs added to the endothelial monolayers and exposed to LPS enhanced the increase . PTX attenuated the permeability increase mediated by LPS-exposed PMNs . PTX did not prevent the LPS-induced increase in permeability when PMNs were not present, although PTX increased endothelial cell cAMP levels . These data demonstrate that 1) PTX does not prevent lung injury in granulocyte-depleted guinea pigs; 2) PTX does not prevent LPS-induced increases in endothelial cell permeability, despite increased cAMP levels; and 3) PTX attenuates PMN-dependent increases in endothelial cell permeability. Appl Environ Microbiol, 1991 Jul, 57(7), 2012 - 5 Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway; Adamowicz M et al.; Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ) . PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate . Aerobically, E . coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E . coli in a defined glucose-salts medium . Thus, for E . coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway . In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor . Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism . Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed . We suggest that E . coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment. J Clin Microbiol, 1991 Jul, 29(7), 1290 - 4 Expression in Escherichia coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna virus strain ZZV 1050: application of recombinant protein in enzyme-linked immunosorbent assay for the detection of caprine and ovine lentiviruses; Zanoni RG et al.; Maedi-visna in sheep and caprine arthritis-encephalitis in goats are caused by two closely related and widespread lentiviruses . The infections are characterized by life-long virus persistence and slow induction of antiviral antibodies . The diagnosis is based on the detection of antiviral antibodies . We have used the polymerase chain reaction (PCR) to amplify a part of the gag gene coding for the entire capsid protein and for parts of the matrix and nucleocapsid proteins . Sequencing of the PCR fragment of the Dutch maedi-visna virus strain ZZV 1050 revealed 85 and 92% homology to the DNA and deduced amino acid sequences, respectively, of the distantly related Icelandic visna virus strain 1514 . The respective homologies with caprine arthritis-encephalitis virus strain CO were 76 and 80% . The PCR fragment was cloned into pGEX-2T and expressed as a glutathione S-transferase fusion protein . The recombinant protein could be detected on immunoblots by using a monoclonal antibody and polyclonal antisera and was further purified by glutathione-based affinity chromatography . Enzyme-linked immunosorbent assay with purified recombinant fusion protein is shown to be a sensitive and specific diagnostic tool for the detection of lentiviral infection in goats and sheep. Mol Gen Genet, 1991 Jul, 227(3), 433 - 7 Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H; Itaya M et al.; The rnh gene of Escherichia coli encodes RNase H . rnh mutants display at least two phenotypes: (1) they require functional RecBCD enzyme for growth; thus rnh-339::cat recB270 (Ts) and rnh-339::cat recC271 (Ts) strains are temperature sensitive for growth; (2) rnh mutants permit replication that is independent of the chromosomal origin, presumably by failing to remove RNA-DNA hybrids from which extra-original replication can be primed . We report here that manifestation of these two phenotypes occurs at different levels of RNase H function; we have examined partially functional rnh mutants for their in vitro RNase H activity, their ability to rescue viability in recB or recC cells and their ability to permit growth of mutants incapable of using oriC {dnaA (Ts)}. Mol Gen Genet, 1991 Jul, 227(3), 424 - 32 A combination of RNase H (rnh) and recBCD or sbcB mutations in Escherichia coli K12 adversely affects growth; Itaya M et al.; Colony forming ability of Escherichia coli strains carrying the rnh-339::cat mutant allele is strongly dependent on the recBCD and sbcB genes . A mutation inactivating either the RecBCD nuclease or exonuclease I (sbcB) is sufficient to restrict severely the efficiency of plating of strains carrying the rnh-339::cat mutation . Combining a non-lethal temperature-sensitive mutation in the RecBCD nuclease, recB270 (Ts) or recC271 (Ts), with rnh-339::cat renders strains temperature sensitive for growth, even though rnh+ strains with the recB270 (Ts) or recC271 (Ts) alleles are viable at 42 degrees C . The recombinational functions of the RecBCD nuclease can be excluded as the source of lethality on the basis of the following observations . Introduction of a recombination proficient, exonuclease defective recD1009 allele or production of the phage lambda GamS protein (an inhibitor of the RecBCD exonuclease activity) in an rnh-339::cat strain dramatically delays or impairs the ability of such strains to form colonies . Restoration of recombination proficiency by inclusion of an sbcB15 mutation with recB21 recC22 mutations does not restore the ability of the rnh-339::cat mutant strains to plate normally . A recBCD+ strain bearing the rnh-339::cat and sbcB15 mutations forms very few visible colonies after 24 h but forms colonies at normal frequencies after 48 h of incubation . Finally, plating efficiencies of strains are unaffected when the RecBCD recombination pathway is inactivated by introduction of recA56 into an rnh-339::cat strain . These results imply that the defective growth of rnh-339::cat recBCD strains is due to a defect in repair and not recombination mediated by either the RecBCD or the RecF pathway. J Clin Pathol, 1991 Jul, 44(7), 609 - 10 Haemorrhagic colitis and haemolytic-uraemic syndrome: false positive reaction with a rotavirus latex agglutination test; Bendall RP et al.; A stool sample from a child with haemorrhagic colitis and haemolytic-uraemic syndrome gave a positive reaction with the RotaScreen latex agglutination test in the absence of other evidence of rotavirus infection . When this test is performed on bloody specimens, positive reactions should be interpreted with caution and confirmed by other means. Biochem J, 1991 Jul 1, 277 ( Pt 1), 89 - 96 Direct assay for O6-methylguanine-DNA methyltransferase and comparison of detection methods for the methylated enzyme in polyacrylamide gels and electroblots; Major GN et al.; We describe in detail a direct assay for the substrate-inactivated DNA-repair enzyme, O6-methylguanine-DNA methyltransferase (O6-MT), which measures the transfer of radiolabelled methyl groups from a prepared O6-methylguanine-DNA substrate to the protein fraction of an enzyme-containing cell/tissue extract . This assay, a modification of a previously suggested method for monitoring O6-ethylguanine-DNA repair {Renard, Verly, Mehta & Ludlum (1983) Eur . J . Biochem . 136, 461-467}, is sensitive, highly reproducible, accurate and is, as described here and relative to previously published methods, well suited for use with a large number of test samples . We identified two problems with the O6-{Me-3H}methylguanine-DNA substrate used in the present work and in other reported assay: firstly, that of progressively higher assay backgrounds with increasing age of substrate, which was nullified by once-only purification of the double-stranded substrate by hydroxyapatite chromatography; secondly, a substrate of high specific radioactivity (30 Ci/mmol), made with freshly prepared tritiated methylnitrosourea, behaved as a substrate of 5 Ci/mmol when referenced against radiolabelled O6-methylguanine-DNA made with either {3H}- or {14C}-methylnitrosourea at the lower specific radioactivities of 1 Ci/mmol and 61 mCi/mmol respectively . This apparently stemmed from the known instability of high-specific-radioactivity {3H}methylnitrosourea and indicated that an expected increase in sensitivity of the assay does not necessarily result from increasing the specific radioactivity of substrates above approx . 1 Ci/mmol . Although O6-MT was stable to preincubation at 25 degrees C, marked losses of activity were observed at 37 degrees C, and more so at 45 degrees C . Enzyme lability at the higher temperatures was not, however, seen during preincubation in the presence of its substrate . O6-{Me-3H}methylguanine-DNA, which apparently protected O6-MT against thermal inactivation . As previously seen with other human cells and tissues, extracts of human spleen in the present study showed wide interindividual differences in O6-MT specific activity (18-fold), which spanned the range 50-900 fmol/mg of protein . Cultured human lymphoblastoid Jurkat cells contained approx . 57,000 enzyme molecules/cell . Substrate-inactivated {Me-3H}methylated O6-MT was analysed by SDS/PAGE and electroblotting . The different but similarly sized forms of this enzyme that we previously detected in human spleen {Major, Gardner, Carne & Lawley (1990) Nucleic Acids Res . 18, 1351-1359} were clearly resolved by fluorography of electroblots, but only at considerable expense of time . As expected, scintillation counting of the protein extracted from gel slices and linear-wire scanning of enzyme-associated radioactivity on electroblots were quicker methods for detecting the {Me-3H}methylated inactivated O6-MT. Clin Exp Immunol, 1991 Jul, 85(1), 66 - 9 Antigen DNA isolated from immune complexes in plasma of patients with systemic lupus erythematosus hybridizes with the Escherichia coli lac Z gene; Terada K et al.; Antigen DNA was isolated from immune complexes in plasma of three patients with active systemic lupus erythematosus (SLE) using affinity column . The antigen DNA thus obtained was subjected to hybridization experiments in order to investigate its origin . Unexpectedly, plasmid pUC18 used as a probe was found to hybridize with the antigen DNA, pUC18 was then cleaved into three fragments with the restriction enzyme HaeII . A 445-bp fragment containing lac Z DNA hybridized with the antigen DNA . Finally, the lacZ DNA itself was found to hybridize with the antigen DNA . These data strongly suggest that the antigen DNA obtained from three patients is of bacterial origin. Mutat Res, 1991 Jul, 249(1), 125 - 33 Induction of gamma delta transposition in response to elevated glucose-6-phosphate levels; Lee AT et al.; The nonenzymatic glycosylation of nucleic acids in vitro by the reducing sugars, glucose or glucose-6-phosphate, alters both physical and biological properties . Recent investigations have demonstrated that elevated intracellular levels of glucose-6-phosphate in glycolytic mutants of E . coli resulted in a concentration-associated increase in mutations of a target plasmid . The majority of the plasmid mutations were due to large (greater than 1 kb) insertions or deletions . We describe here the further analysis of mutant plasmids isolated from bacteria grown under conditions which were conducive to the intracellular accumulation of glucose-6-phosphate . We have found that a number of the insertional plasmid mutations were the result of the movement of the transposable element gamma delta from the host genome into the plasmid . The frequency of gamma delta transposition was also associated with the amount of glucose-6-phosphate accumulated in the bacterial cells . Furthermore, the presence of another transposable element, either Tn 5 or Tn 10 in the host genome increased the rate of gamma delta transposition without affecting its own movement . The observed increase in gamma delta transposition suggests a novel mechanism of induction by reducing sugars which may be the result of DNA modifications by reducing sugars. J Bacteriol, 1991 Jul, 173(14), 4474 - 81 Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S; Lange R et al.; The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli . Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger . Size reduction of E . coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells . Stationary-phase induction of bolA is dependent on sigma S . The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes . However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5867 - 71 Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1; Gu MX et al.; We have isolated a cDNA encoding a third type of protein-tyrosine-phosphatase . We screened human megakaryoblastic cell line (MEG-01) an umbilical vein endothelial cell cDNA libraries to obtain a 3.7-kilobase cDNA designated PTPase MEG . Northern blot analysis of MEG-01 RNA detected a 3.7-kilobase transcript, suggesting that a full-length cDNA has been identified . PTPase MEG cDNA contains an open reading frame of 926 amino acids . The cDNA has a G+C-rich 5' untranslated region of 771 nucleotides that has the potential to form stable stem-loop structures and has two upstream ATG codons . The predicted protein (Mr = 105,910) has no apparent membrane-spanning region and contains a single protein-tyrosine-phosphatase domain (amino acids 659-909) that is 35-40% identical to previously described tyrosine-phosphatase domains . The recombinant phosphatase domain possesses protein-tyrosine-phosphatase activity when expressed in Escherichia coli . The amino-terminal region (amino acids 31-367) is 45% identical to the amino terminus of human erythrocyte protein 4.1, a cytoskeletal protein . The identification of a protein-tyrosine-phosphatase that is related to cytoskeletal proteins implies that cell signaling activities reside not only in transmembrane receptors but in cytoskeletal elements as well. J Bacteriol, 1991 Jul, 173(13), 4116 - 23 Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS; Kawula TH et al.; Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter . DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS . Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process . This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles . One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter . The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature . We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method . Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion. J Bacteriol, 1991 Jul, 173(13), 4039 - 48 cis-acting ompF mutations that result in OmpR-dependent constitutive expression; Slauch JM et al.; OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium . We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity . Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility . These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region. J Bacteriol, 1991 Jul, 173(13), 3958 - 65 Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function; Brandner JP et al.; Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides . The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm . To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase . All five of the fusion proteins are exported to the periplasmic space . The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment . Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers . Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R . sphaeroides . Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A . R . Varga and S . Kaplan, J . Bacteriol . 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier. J Bacteriol, 1991 Jul, 173(13), 3949 - 57 Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides; MacGregor BJ et al.; Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain . cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically . We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension . Complementation of a cycA null mutant shows that there are at least two cycA promoters: one within 89 bp upstream of the translation initiation codon for a transcript beginning at -28, and at least one within 484 bp upstream for the remaining nine 5' ends . The 5' ends at -28 and -137 are more abundant in aerobically grown cells, while those at -38, -155, -250, and -300 are more abundant photosynthetically . DNA sequences with homology to the Escherichia coli sigma 70 consensus promoter sequence precede the 5' ends at -28 and -274, and there is weak homology upstream of the -82 and -250 ends. J Infect Dis, 1991 Jul, 164(1), 22 - 8 Analysis of antibody titers to Epstein-Barr virus nuclear antigens in sera of patients with Sjögren's syndrome and with rheumatoid arthritis; Inoue N et al.; To examine if Epstein-Barr virus (EBV) infection is associated with the two autoimmune diseases, Sjogren's syndrome and rheumatoid arthritis, IgG antibody titers of sera from patients with the disorders were evaluated for five constituents of EBV nuclear antigens (EBNA-1, -2, -3, -4, and -6) . To circumvent interference by autoantibodies in the sera, fusion proteins synthesized in Escherichia coli were used as specific antigens . By ELISA the average IgG antibody titers to domains of the five EBNAs, especially the amino-terminal domain of EBNA-2, in sera of patients with Sjogren's syndrome were slightly higher than those in normal sera . The tendency of sera from Sjogren's syndrome patients to have higher reactivities to the EBNA domains was also observed by immunoblotting . By comparison, few heightened serologic responses to EBNAs were observed in sera from patients with rheumatoid arthritis . Further analyses are required to determine if EBV is associated with Sjogren's syndrome in any way. Circ Res, 1991 Jul, 69(1), 12 - 25 Priming by platelet-activating factor of endotoxin-induced lung injury and cardiovascular shock; Rabinovici R et al.; Platelet-activating factor (PAF) is a glycerophospholipid known for its unusual potent vasoactive and proinflammatory activities . The present study examined whether PAF might serve as a priming factor in endotoxin-induced tumor necrosis factor-alpha (TNF alpha) synthesis, cardiovascular shock, and lung injury in anesthetized rats . Intravenous infusion of PAF (1 pmol/kg/min for 60 minutes, n = 5) alone or endotoxin (0.1 micrograms/kg i.v . bolus, n = 5) failed to alter blood pressure, serum TNF alpha and thromboxane B2, platelet and leukocyte count, and hematocrit, nor was lung histology, myeloperoxidase activity, and water content changed . In contrast, the combined administration of PAF and endotoxin markedly elevated serum TNF alpha (1,359 +/- 362 pg/ml, n = 5, p less than 0.01) and thromboxane B2 (43 +/- 5 pg/100 microliters, n = 8, p less than 0.01) along with hypotension, hemoconcentration, leukopenia, and thrombocytopenia . Most notably, the combined regimen caused neutrophil aggregation, adhesion, and accumulation into the lung parenchyma along with platelet-fibrin deposits in postcapillary venules, pulmonary edema, and increased lung myeloperoxidase activity . The role of PAF in this process was confirmed by 1) the prevention of the priming effect by pretreatment with the PAF antagonist BN 50739 (n = 5), and 2) the failure of lyso-PAF, the cardinal nonactive PAF-metabolite, to prime for endotoxin-induced production of TNF alpha (n = 4) . These data suggest that PAF could serve as a key mediator in priming for endotoxin-induced tissue injury, especially the typical pulmonary pathophysiology of adult respiratory distress syndrome, a severe pathological outcome of septic shock, burns, and multiple organ injury. Agric Biol Chem, 1991 Jul, 55(7), 1817 - 22 Efficient production of Taka-amylase A by Trichoderma viride; Cheng C et al.; An efficient heterologous protein production system was developed in Trichoderma viride, a very efficient cellulase producer . An expression vector containing the Taka-amylase A gene from Aspergillus oryzae, which was fused to the strong promoter and signal peptide sequence of the cellobiohydrolase 1 gene (cbh1) of T . viride, and the hygromycin B resistance gene was used to transform protoplasts of T . viride . Using hygromycin B resistance, a frequency of 3 transformants per microgram DNA on average was obtained . One transformant showed highly elevated alpha-amylase production, 1.0 g/l, which was shown to be under the control of the cbh1 gene promoter . Analysis of the chromosomal DNA of the transformant showed the integration of more than one copy of the vector. J Biotechnol, 1991 Jul, 19(2-3), 159 - 72 Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and relA+ strains; van Bakel M et al.; To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period . Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared . To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4 degrees C, with and without previous glucose starvation of the cells . It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes . In addition, upon restoration of maximal growth rate at 37 degrees C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h . Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage . Recovery of the starved cells was most rapid in the relA+ strain which, contrary to the isogenic relA strain, showed no overshoot and reached its final steady-state size within 2 h. Biochemistry, 1991 Jun 18, 30(24), 5955 - 63 DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3; Benevides JM et al.; The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif . We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102) . Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides . Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding . Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups . These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family . These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation . The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes . However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex . The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution . This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions. Biochem Biophys Res Commun, 1991 Jun 28, 177(3), 1319 - 24 The smg GDS-induced activation of smg p21 is initiated by cyclic AMP-dependent protein kinase-catalyzed phosphorylation of smg p21; Itoh T et al.; We have shown that smg p21B is phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and that membrane acidic phospholipids such as phosphatidic acid and phosphatidylinositol markedly inhibit the smg GDS-induced activation of smg p21B . However, we show here that phosphatidic acid and phosphatidylinositol exhibit a less inhibitory effect on the smg GDS-induced activation of the phosphorylated form of smg p21B . Thus, in the presence of membrane acidic phospholipids, the smg GDS-induced activation of smg p21B is totally dependent on the protein kinase A-catalyzed phosphorylation of smg p21B . Since smg p21B is located on the membranes in resting cells, it is likely that the smg GDS-induced activation of smg p21B is initiated by the protein kinase A activation. Cell, 1991 Jun 28, 65(7), 1233 - 42 The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo; Lin-Chao S et al.; We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo . Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5 . A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication . Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype . RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number. J Biol Chem, 1991 Jun 15, 266(17), 11044 - 50 Cloning, sequencing, and characterization of Escherichia coli thioesterase II; Naggert J et al.; The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced . A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide . The recombinant enzyme constituted up to 25% of the soluble proteins of E . coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da . Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da . The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins . Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate . The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested . E . coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA . This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E . coli . Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E . coli at any stage of cell growth and the physiological role of this enzyme remains an enigma. Nucleic Acids Res, 1991 Jun 25, 19(12), 3431 - 4 Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene; Hu J et al.; The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively {Hu, J . and Bogorad, L . (1990) Proc . Natl . Acad . Sci . USA . 87, pp . 1531-1535} . These genes have segments that specify amino acid sequences homologous to those of E . coli RNA polymerase subunits . The plastid gene products are designated b", b and a, respectively . We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E . coli rpoC gene . Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase . This polypeptide is designated subunit b' . Three polypeptides unrelated to RNA polymerase have also been identified in this preparation. Nucleic Acids Res, 1991 Jun 25, 19(12), 3337 - 43 Site directed substitution of 5-hydroxymethyluracil for thymine in replicating phi X-174am3 DNA via synthesis of 5-hydroxymethyl-2'-deoxyuridine-5'-triphosphate; Levy DD et al.; 5-hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of Thy . It is removed from DNA by HmUra-DNA glycosylase . To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairability of HmUra, a HmUra residue was substituted for Thy in a target (amber) codon by in vitro extension of an oligonucleotide primer annealed to phi X-174am3 virion DNA . This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension . E . coli spheroplasts were transfected with the HmUra-containing DNA and the yield of revertant phage determined following replication in the bacterial host . Since E . coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed in the absence of repair . chi 2c analysis showed that replacing Thy with HmUra did not result in an increase in revertant phage . These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis. Nucleic Acids Res, 1991 Jun 25, 19(12), 3319 - 23 Stability of DNA thymine hydrates; Ganguly T et al.; Pyrimidine hydrates are products of ultraviolet irradiation of DNA . We have already demonstrated the formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in irradiated poly(dA-dT):poly(dA-dT) . These are released from DNA as free bases by bacterial or human glycosylases . Thymine hydrate stabilities were studied in irradiated DNA substrates using purified E . coli endonuclease III as a reagent for their removal . After irradiation, substrate poly(dA-dT):poly(dA-dT), radiolabeled in thymine, was incubated at 50, 60, 70 or 80 degrees C, cooled, and then reacted with the enzyme under standard conditions . Thymine hydrates were assayed by enzymic release of labeled material into the ethanol-soluble fraction . Their identities were confirmed by high performance liquid chromatography . The decay of thymine hydrates in heated DNA followed first-order kinetics with a k = 2.8 x 10(-5)/sec at 80 degrees C . These hydrates were also detected in lesser quantities in the unirradiated, control substrate . Extrapolation from an Arrhenius plot yields an estimated half-life of 33.3 hours at 37 degrees C for DNA thymine hydrates . Such stability, together with their formation in unirradiated DNA, suggest thymine hydrates to be formed under physiological conditions and to be sufficiently stable in DNA to be potentially genotoxic . This necessitates their constant removal from DNA by the excision-repair system. Biochemistry, 1991 Jun 25, 30(25), 6322 - 9 Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro; Li YF et al.; DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer . The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor . During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity . Previous nanosecond flash photolysis studies {Heelis, P.F., Okamura, T., & Sancar, A . (1990) Biochemistry 29, 5694-5698} indicated that excitation of FADH . either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH . quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme . Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction . We found that W306F mutation abolished photoreduction of FADH . without affecting the excited-state properties of FADH . or the substrate binding (KA approximately 10(9) M-1) of the enzyme . The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH . is an essential step for photorepair by photolyase in vitro . Chemical reduction of FADH . of the mutant enzyme restored the specificity constant to the wild-type level. Biochemistry, 1991 Jun 25, 30(25), 6241 - 6 Fluorescence analysis of tryptophan-containing variants of the LamB signal sequence upon insertion into a lipid bilayer; McKnight CJ et al.; To investigate the interaction of the LamB signal sequence with lipid bilayers, we have synthesized three tryptophan-containing analogues of the wild-type signal peptide . The tryptophan residues were used as intrinsic fluorescent probes of the N-terminal (position 5), central (position 18), and C-terminal (position 24) regions of the 25-residue peptide . The tryptophan substitutions did not significantly alter the physical properties of the wild-type signal peptide . In the presence of lipid vesicles which mimic the composition of the Escherichia coli inner membrane, the peptides adopt alpha-helical structure, and the tryptophan fluorescence emission maximum is shifted to shorter wavelength, indicating that the peptides insert into the acyl chain region of the lipid bilayer . Fluorescence quenching by soluble, aqueous-phase (I-), and membrane-resident (nitroxide-labeled lipids) quenchers was used to locate the tryptophans in each peptide within the bilayer . The C-terminus was interfacial while the central region of the signal sequence was deeply buried within the acyl chain region of the bilayer . The tryptophan at position 5 was buried but less deeply than the tryptophan at position 18 . This topology is consistent with either a looped or a transmembrane orientation of signal peptide . However, either structure must accommodate the high helical content of the peptides in vesicles . These results indicate that the LamB signal sequence spontaneously inserts into the acyl chain region of lipid membranes in the absence of any of the proteins involved in protein secretion. Biochemistry, 1991 Jun 25, 30(25), 6124 - 7 Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase; Bradley M et al.; The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione {N1,N8-bis(glutathionyl)spermidine} by site-directed mutagenesis of two residues . The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore . The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T . congolense trypanothione reductase, and the properties of substrate analogues of trypanothione . Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion. J Biol Chem, 1991 Jun 25, 266(18), 12090 - 8 Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication; Erdile LF et al.; Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells . RP-A is essential for SV40 DNA replication and may also be important in genetic recombination . The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported . The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae . Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type . The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A . The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex . The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template . However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits. J Biol Chem, 1991 Jun 25, 266(18), 12053 - 7 Cloning and expression of functional rabbit muscle creatine kinase in Escherichia coli . Addressing the problem of microheterogeneity; Chen LH et al.; The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli . The amino terminus, specific activity, and electrophoretic mobility of the E . coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle . Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material . The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications . This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes . Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK. J Biol Chem, 1991 Jun 25, 266(18), 12048 - 52 Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll; Kohorn BD et al.; The mechanism by which a protein integrates posttranslationally into a membrane can involve the composition of the membrane itself, domains within the inserting polypeptide, and a number of associating proteins . Some integral membrane proteins do not accumulate to normal levels when certain pigments are deficient, and this has been interpreted to mean that such proteins may be rapidly degraded when not in a correct complex . Alternatively, pigments could facilitate the movement of some proteins from an aqueous to a lipid environment . To determine whether chlorophyll is absolutely required for the membrane integration of the light-harvesting chlorophyll-binding protein (LHCP) of chloroplast thylakoid membranes, we have expressed LHCP in Escherichia coli that lacks photosynthetic pigments . LHCP is targeted to the bacterial inner membrane by the addition of a bacterial signal peptide and cannot be extracted from these membranes by NaOH, NaBr, or Na2HCO3 but is extracted by 0.2% Triton X-100 . Treatment of isolated right-side-out and inside-out bacterial inner membrane vesicles with trypsin reveals that only the amino terminus of LHCP is exposed on the cytoplasmic face, and the remaining portion of the protein is inaccessible . Treatment of the inside-out vesicles with trypsin followed by alkaline extraction shows that LHCP is intrinsic to the membrane and is not anchored solely by the bacterial signal peptide . Chlorophyll, therefore, is not required for LHCP to integrate into a membrane, but in the absence of these pigments this process is observed to be inefficient. J Biol Chem, 1991 Jun 25, 266(18), 11766 - 73 Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli; Koehler DR et al.; An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli . The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W . (1987) J . Mol . Biol . 198, 187-202) . On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed . A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand . This bias is reduced in an excision repair deficient (UvrB-) strain . In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand . This bias is also markedly reduced in the UvrB- strain . In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C . (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E . coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis. J Biol Chem, 1991 Jun 25, 266(18), 11718 - 25 Recombinant rhizopuspepsinogen . Expression, purification, and activation properties of recombinant rhizopuspepsinogens; Chen Z et al.; A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg) . Recombinant Rpgs which were expressed in the cytosol of E . coli as inclusion bodies (cRpg and tRpg) were not active . After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity . The third zymogen, pRpg, which was secreted to the periplasmic space of E . coli with an omp leader, was fully active and also was purified . The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture) . Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively . tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture . This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction . All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5 . Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis . At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens . For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2 . The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively . The activation rate decreased as pH was raised above pH 2 . At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place . Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen . These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen . Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion. J Biol Chem, 1991 Jun 25, 266(18), 11789 - 96 Mutations that affect the folding of ribose-binding protein selected as suppressors of a defect in export in Escherichia coli; Teschke CM et al.; It has been proposed (Randall, L . L., and Hardy, S . J . S . (1986) Cell 46, 921-928) that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export . As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export . (Liu, G., Topping, T . B., Cover, W . H., and Randall, L . L . (1988) J . Biol . Chem . 263, 14790-14793) . Here we describe mutations of the gene encoding ribose-binding protein that were selected as suppressors of a defect in export of that protein and that alter the folding pathway . We propose that selection of such suppressors may provide a general method to obtain mutations that affect the folding properties of any protein that can be expressed and exported in E . coli. Nucleic Acids Res, 1991 Jun 25, 19(12), 3251 - 3 Polyinosinic acid as a carrier in the microscale purification of total RNA; Winslow SG et al.; Three different RNA carriers were compared for use in microscale RNA isolation and subsequent cDNA synthesis and amplification via the polymerase chain reaction . E.coli rRNA alone gave considerable cDNA synthesis which under standard carrier conditions overwhelmed cDNA synthesis from lymphocyte mRNA . Yeast tRNA caused inhibition of mRNA primed cDNA synthesis, giving low levels of cDNA synthesis when used without cellular RNA . In contrast, commercially available poly I alone did not prime detectable cDNA synthesis nor did it inhibit such synthesis primed by cellular mRNA . When RNA preparations were made using these three carriers and decreasing numbers of starting lymphocytes, poly I allowed the detection of cDNA from two orders of magnitude fewer lymphocytes than the other carriers . Thus poly I was found to be a superior carrier molecule for microscale RNA preparations suitable for reverse transcription and subsequent amplification using the polymerase chain reaction. Biochemistry, 1991 Jun 25, 30(25), 6210 - 6 Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system; Cho HJ et al.; A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization . To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized . Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release . The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM. Biochemistry, 1991 Jun 25, 30(25), 6135 - 41 Investigation of the mechanism of phosphinothricin inactivation of Escherichia coli glutamine synthetase using rapid quench kinetic technique; Abell LM et al.; A number of slow tight-binding inhibitors are known for glutamine synthetase that resemble the geometry of the tetrahedral intermediate formed during the enzyme-catalyzed condensation of gamma-glutamyl phosphate and ammonia . One of these inhibitors, phosphinothricin {L-2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid}, has been investigated by rapid kinetic methods . Phosphinothricin not only exhibits the kinetic properties of a slow tight-binding inhibitor but also undergoes phosphorylation during the course of the ATP-dependent inactivation . The acid lability of phosphinothricin phosphate enabled investigation of the kinetics of glutamine synthetase inactivation using rapid quench kinetic techniques . The rate-limiting step in the inhibition reaction is the binding of inhibitor (0.004-0.014 microM-1 s-1) and/or a conformational change associated with binding, which is several orders of magnitude slower than the binding of ATP . The association rate of phosphinothricin depends on which metal ion is bound to the enzyme (Mn2+ or Mg2+) . With Mn2+ bound to glutamine synthetase the rate of association and the phosphorylation rate are faster than when Mg2+ is bound . The data are interpreted with use of a model in which the binding of a substrate analogue with a tetrahedral moiety enhances the phosphorylation rate of the reaction intermediate; however, the initial binding interaction is retarded because the enzyme has to bind a molecule that has a "transition-state" geometry rather than a ground-state substrate structure . During the course of the inactivation, progressively slower rates for binding and phosphoryl transfer were observed, indicating communication between active sites. Nucleic Acids Res, 1991 Jun 25, 19(12), 3395 - 402 Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7; Tang Y et al.; Tn7, a large bacterial transposon encodes 5 proteins required for its transposition . We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain . This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration . DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected . Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein . ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins . Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix. J Biol Chem, 1991 Jun 25, 266(18), 11621 - 7 Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate; Kanaya S et al.; The handle region (residues 84-99) in ribonuclease HI (RNase HI) from Escherichia coli, which is rich in basic amino acid residues, was altered by alanine-scanning mutagenesis . Fifteen mutant proteins were purified to homogeneity and analyzed for the enzymatic activity . A mutation of either of 2 tryptophan residues at 85 or 90 resulted in a large increase in the Km value along with a large decrease in the Vmax value . These values probably resulted from conformational changes introduced by the mutations as indicated by the CD spectra of these mutant proteins . All other mutant enzymes had Vmax values similar to that of the wild-type enzyme . In contrast, replacement of any basic amino acid residue in the handle region, except for lysine 86, yielded proteins whose Km values were 3-5-fold higher than the wild-type enzyme . Such effects were shown to be cumulative, suggesting strongly that the cluster of positive charges in the handle region is important for the effective binding of the substrate . Interestingly, the region of human immunodeficiency virus reverse transcriptase with homology to E . coli RNase HI lacks the handle region which may account for the poor RNase H activity of the domain when separated from the polymerase domain. FEBS Lett, 1991 Jun 24, 284(2), 270 - 2 Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli; Kawata Y et al.; Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method . The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph . The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit . The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis. FEBS Lett, 1991 Jun 24, 284(2), 178 - 83 Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin; Chandrasekhar K et al.; As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed . The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra . The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra . The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin. Biochim Biophys Acta, 1991 Jun 24, 1078(2), 199 - 207 An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli; Bobst EV et al.; A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported . Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer . Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl) . The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM . It is, therefore, metastable and forms only under reverse titration conditions in low NaCl . The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex . The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V . and Ireland, J.C . (1982) J . Biol . Chem . 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C . and Bobst, E.V . (1984) J . Biol . Chem . 259, 2130) by EPR competition assays . In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein . The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n. Proc R Soc Lond B Biol Sci, 1991 Jun 22, 244(1311), 211 - 7 Iron (III) can be transferred between ferritin molecules; Bauminger ER et al.; The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite . The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize . Here we present evidence, from Mossbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules . Experiments were done with both horse spleen ferritin and recombinant human ferritin . Mossbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron . In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h) . Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time . Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites . In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters . In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters . Such migration must now be taken into account in any model of ferritin iron-core formation. J Mol Biol, 1991 Jun 20, 219(4), 747 - 55 Spatial arrangement of sigma-factor and core enzyme of Escherichia coli RNA polymerase . A neutron solution scattering study; Lederer H et al.; By means of neutron solution scattering we determined the position and orientation of core enzyme and sigma-factor within the Escherichia coli RNA polymerase holoenzyme with the aim of improving existing models . The individual components, core enzyme (E) and sigma-factor (sigma), were highlighted by deuterium labeling and their center-to-center distances determined in the monomeric and the dimeric holoenzyme . The following distance parameters were obtained: dE1-sigma 1 = 8.6(+/- 1) nm, dE1-E2 = 11.5(+/- 1) nm, d sigma 1-sigma 2 = 12.0(+/- 0.7) nm, dE1-sigma 2 = 9(+/- 3) nm . Using a triangulation procedure the position of the sigma-factors, sigma 1 and sigma 2, were determined with respect to the mass center of the core enzyme molecules, E1 and E2, assuming a symmetrical arrangement of the holoenzyme molecules in the dimer (C2 symmetry) . In addition, the orientation of the sigma-factor with respect to core enzyme was estimated by means of model calculations . The obtained model of holoenzyme depicts the sigma-factor as buried in a groove of core enzyme, probably between the large subunits beta' and beta. J Mol Biol, 1991 Jun 20, 219(4), 645 - 54 The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments; Dutreix M et al.; Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important . We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange . When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends . Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament . These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur . Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo. J Mol Biol, 1991 Jun 20, 219(4), 615 - 22 DNA looping alters local DNA conformation during transcription; Wu HY et al.; The effect of protein-mediated DNA looping on local DNA conformation during active transcription was studied using the lac repressor-operator system . Our results suggest that lac repressor-mediated DNA looping within a plasmid DNA molecule containing two lac repressor binding sequences in vivo effectively separates plasmid DNA into two topological domains . Supercoils generated by transcription within each topological domain can be rapidly removed by DNA topoisomerase I. J Mol Biol, 1991 Jun 20, 219(4), 593 - 4 Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12; Delbaere LT et al.; Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system . Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding . The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59% . The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure. J Mol Biol, 1991 Jun 20, 219(4), 623 - 34 In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator; Khoury AM et al.; Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits . Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues . Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies . Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically . To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter . In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment . These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4) . The expression of these lac repressor fragments in the cell was verified by radioimmunoassays . Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo . These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain. J Mol Biol, 1991 Jun 20, 219(4), 655 - 63 Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor; Drexler K et al.; The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor . This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virion's long tail fibers . Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA . Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area . Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111 . It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX . Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X . This phage could not grow on a mutant of E . coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain . It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues . Selection for resistance to them in E . coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type . Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12 . By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38 . All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38 . In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA . It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide. Nature, 1991 Jun 20, 351(6328), 624 - 9 Crystal structure of an N-terminal fragment of the DNA gyrase B protein; Wigley DB et al.; The crystal structure of an N-terminal fragment of the Escherichia coli DNA gyrase B protein, complexed with a nonhydrolysable ATP analogue, has been solved at 2.5 A resolution . It consists of two domains, both containing novel protein folds . The protein fragment forms a dimer, whose N-terminal domains are responsible for ATP binding and hydrolysis . The C-terminal domains form the sides of a 20 A hole through the protein dimer which may play a role in DNA strand passage during the supercoiling reaction. Int J Cancer, 1991 Jun 19, 48(4), 623 - 30 Production of a recombinant human T-cell leukemia virus type-I trans-activator (tax1) antigen and its utilization for generation of monoclonal antibodies against various epitopes on the tax1 antigen; Tanaka Y et al.; A 42-kDa recombinant protein, PX141, consisting of the trans-activator protein encoded by human T-cell leukemia virus (HTLV-1) (tax1 antigen) and the amino-terminal fusion peptide of 12 amino acid residues of the alpha-peptide encoded by the plasmid pUC19 was produced . In order to investigate the immunogenicity of the tax1 antigen, mice were immunized with the purified PX141 and 4 anti-tax1 monoclonal antibodies (MAbs) designated TAXY-1, TAXY-6, TAXY-7 and TAXY-8 were generated, and their reactivity was characterized along with another anti-tax1 MAb, Lt-4 . Immunoblot assays showed that all the MAbs reacted with the PX141, the native tax1 antigen expressed in various HTLV-1-infected cell lines and the gp68 of MT-2 cells expressing the tax1 amino acids 94-353 . Immunoblot assays using recombinant, truncated tax1 antigens, XD59 (expressing amino acids 180-338) and XD128 (expressing amino acids 1-47 and 286-353) showed that: (1) TAXY-1 and Lt-4 did not react with either antigen; (2) TAXY-6 and TAXY-8 reacted with only XD128: and (3) TAXY-7 reacted with both . In addition, TAXY-1, but not the other MAbs, reacted with a putative tax antigen of an STLV-I-infected cell line, designated RfM26-I . Competitive binding assays showed that TAXY-6 and TAXY-8 did not compete against each other . Sera from HTLV-I-infected humans interfered with the binding of all of these anti-tax1 MAbs . These results indicate that the tax1 antigen and the PX141 express at least 5 distinct epitopes recognized by human and mouse antibodies. Biochemistry, 1991 Jun 18, 30(24), 5927 - 34 Reaction of indole and analogues with amino acid complexes of Escherichia coli tryptophan indole-lyase: detection of a new reaction intermediate by rapid-scanning stopped-flow spectrophotometry; Phillips RS; The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry . Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation . Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine . Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 512 nm and the appearance of a new peak at 345 nm . Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm . Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm . This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan . This new intermediate is formed faster than catalytic turnover (kcat = 6.8 s-1) and may be an alpha-aminoacrylate intermediate bound as a gem-diamine. Biochemistry, 1991 Jun 18, 30(24), 5851 - 7 Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone; McManus S et al.; Two-dimensional (2D) 1H NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody Fv fragment expressed in Escherichia coli . Point mutations of residues in the CDR loops of the Fv fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals . Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain . The benzyl portion of 2-phenyloxazolone is located between these two residues . The binding site is close to the surface of the Fv fragment . Comparison with a different anti-2-phenyloxazolone antibody, the crystal structure of which has recently been solved, shows that the general location of the hapten-binding site in both antibodies is similar . However, in the crystallographically solved antibody, the hapten is bound farther from the surface in a pocket created by a short CDR3 loop of the heavy chain . In the binding site identified in the Fv fragment studied in this report, this space is probably filled by the extra seven residues of the CDR3. Biochemistry, 1991 Jun 18, 30(24), 5832 - 8 Kinetic and thermodynamic characterization of the interaction between Q beta-replicase and template RNA molecules; Werner M; The specific binding of the RNA polymerase Q beta-replicase to some of its RNA template molecules, the single-stranded RNA variant MDV and also Q beta-RNA, was studied under various conditions by using a gel-retardation assay as well as filter retention . The dissociation of the replicase-RNA complex proceeds with first-order kinetics . The dependence of the dissociation rate constant on the concentration of monovalent ions suggests that there are three contacts between the midivariant (MDV) RNA and the replicase . Through analysis of the temperature dependence of the dissociation rate constant, values of 35 and 43 kJ/mol were obtained for the activation energies of complex dissociation between Q beta-replicase and the minus (-) and plus (+) strands of MDV, respectively . The bimolecular association is of second order with high rate constants that increase when the temperature is raised and decrease at higher salt concentrations . The equilibrium constants vary between 4.10(11) M-1 and 5.10(7) M-1, according to the reaction conditions . The temperature dependence of Ka gives delta H = -39 kJ/mol for MDV- and -47 kJ/mol for MDV+ . Under nearly all conditions, distinct differences in the association and dissociation rates of plus and minus strands of MDV are observed . The binding of the small variant MDV to Q beta-replicase is three orders of magnitude stronger than the binding of the natural template Q beta-RNA. Eur J Pharmacol, 1991 Jun 18, 199(1), 115 - 8 Mechanisms of ex vivo aortic hypocontractility in endotoxemic rat; Wakabayashi I et al.; To clarify the mechanism of the vascular hypocontractility in endotoxemia, the effect of endotoxin injection on phosphatidylinositol turnover and the contractile responses to NH4Cl and okadaic acid were investigated in aorta dissected from rats . The basal level of phosphatidylinositol hydrolysis and the phenylephrine- and 5-hydroxytryptamine-stimulated increase in hydrolysis were all markedly reduced in endotoxemic aortas as compared to in control aortas . Stimulation with KCl did not increase phosphatidylinositol hydrolysis in control or endotoxemic aortas . The NH4Cl-induced contractile response was significantly diminished in endotoxemic aorta, whereas the okadaic acid-induced contractile response was not altered . These results suggest that both transmembrane and intracellular signal transduction mechanisms are impaired in the endotoxemic artery whereas the contractile machinery remains intact. Biochemistry, 1991 Jun 18, 30(24), 6036 - 47 Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy; Yamazaki T et al.; The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented . To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied . The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N . Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks . In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C . This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins . Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported. Biochim Biophys Acta, 1991 Jun 17, 1058(2), 107 - 12 Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli; Hibino T et al.; The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli . The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein . The purified precursor apo-plastocyanin was processed to the mature apo-plastocyanin by chloroplast extracts . N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature plastocyanin that was deduced from the nucleotide sequence . The copper could be incorporated into the apo-plastocyanin of mature size in vitro, but could not into the precursor apo-plastocyanin under the same conditions . Absorption spectra and reduction potential of the reconstituted mature plastocyanin were indistinguishable from those of the purified spinach plastocyanin . The electron transfer activities of the reconstituted plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach plastocyanin. Biochim Biophys Acta, 1991 Jun 17, 1058(2), 304 - 11 Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli; Muneyuki E et al.; The hydrolysis of 0.3 microM {alpha,gamma-32P}ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate . The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate . Under all conditions examined, only 10-20% of the {alpha,gamma-32P}ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP . These features differ from those observed with the mitochondrial F1-ATPase . The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate . Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D . and Senior, A.E . (1989) J . Biol . Chem . 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic . Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate . The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E . coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently. Biochem J, 1991 Jun 15, 276 ( Pt 3), 817 - 23 Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein; Mandrup S et al.; A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed . The synthetic gene was assembled from eight partially overlapping oligonucleotides . Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast . The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E . coli . In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein . A fraction highly enriched for recombinant ACBP was obtained by extracting induced E . coli cells with 1 M-acetic acid . Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c . Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group . The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase . It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding. Biochem J, 1991 Jun 15, 276 ( Pt 3), 637 - 41 Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells; Craig FF et al.; Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells . The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells . At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin . At 0.1 mM, the difference between luciferin and the esters was decreased . The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition . The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain . The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants . Alternative luciferin derivatives may allow further improvements in sensitivity. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5212 - 6 In vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana; Kertbundit S et al.; Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants . This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome . The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence . Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA . Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences . After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively . Several different patterns of tissue-specific beta-glucuronidase expression were identified . The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced . After introduction in A . thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated . These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5072 - 6 A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside; Baim SB et al.; LAP267 is a lacI activator protein (LAP) containing an insertion of the transcriptional activation domain of the herpes simplex virus virion protein 16 within the inducer-binding and dimerization domain of the lac repressor protein . LAP267 strongly induces expression in a conditional manner from a minimal simian virus 40 early promoter linked to lac operator sequences . LAP267 is temperature-sensitive, activating expression at 32 degrees C but not at 39.5 degrees C . It is allosterically regulated in a manner opposite that of wild-type lac repressor, in that LAP267 activity is rescued at the nonpermissive temperature by isopropyl beta-D-thiogalactopyranoside (IPTG) . Stable mouse cell lines containing both the LAP267 gene and a LAP-inducible chloramphenicol acetyltransferase (CAT) reporter gene were readily established and exhibited up to a 1200-fold increase in CAT activity within 24 hr upon addition of IPTG . Thus, LAP267 is a powerful inducible switch in mammalian cells, imparting a regulatory stringency similar to that observed with lac repressor in Escherichia coli.
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