Escherichia coli

J Cell Biol, 1990 Dec, 111(6 Pt 1), 2537 - 42
Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection; Hinrichsen R et al.; The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence . Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically . A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium . Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity . However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule . Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

Jpn J Cancer Res, 1990 Dec, 81(12), 1259 - 64
Inhibition of avian myeloblastosis virus reverse transcriptase by aurochloric acid; Semba M et al.; We investigated the inhibitory effects of aurochloric acid (AuCl4H) on reverse transcriptase (RT) derived from avian myeloblastosis virus and DNA polymerase alpha (pol . alpha) purified from HeLa S3 cells . The activities of RT, pol . alpha and E . coli DNA polymerase I (pol . I) with dTTP as the substrate were inhibited 50% at AuCl4H concentrations of 18 microM, 43 microM and 230 microM, respectively . AuCl4H inhibited RT activity competitively with respect to the substrate, dTTP, and uncompetitively with the template/primer, (rA)n(dT)12-18 . In assays with dGTP as the substrate, 50% inhibitions of RT, pol . alpha and pol . I activities were observed at AuCl4H concentrations of 100 microM, 450 microM and 580 microM, respectively . AuCl4H inhibited RT activity uncompetitively with respect to the substrate, dGTP, and noncompetitively with the template/primer, (rC)n(dG)12-18 . AuCl4H at concentrations causing more than 50% inhibition of RT activity had little inhibitory effect on the colony-forming ability of HeLa cells or their syntheses of DNA, RNA and protein.

Arch Biochem Biophys, 1990 Dec, 283(2), 311 - 7
Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein; Ma Q et al.; A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase {NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)} mRNA was expressed in Escherichia coli . The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR) . Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction . The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites . E . coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor . The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M . After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase . The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis . It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase . The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm . Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme . Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid . The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.

J Cell Biol, 1990 Dec, 111(6 Pt 2), 3049 - 64
Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro; Coulombe PA et al.; To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins . Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro . Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency . Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly . Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo . In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation . For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype . Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred . Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

EMBO J, 1990 Dec, 9(13), 4511 - 7
The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence; Zopf D et al.; The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking . Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain) . Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities . Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle . Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19 . In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA . This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA . Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54 . These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9966 - 70
Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site; Whiting P et al.; Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes . Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing . Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions . Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain . Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon . Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site . Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated . Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site . This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes.

Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9519 - 23
Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli; Gasser CS et al.; cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus . In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides . RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested . Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B . napus . A vector was constructed for expression of the tomato cDNA in E . coli . SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band . While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells . This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.

Dev Biol, 1990 Dec, 142(2), 346 - 59
Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene; Gan L et al.; The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm . To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c . All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions . The Spec-lacZ reporter gene plasmids were microinjected into eggs of S . purpuratus, Lytechinus variegatus, and L . pictus, and beta-galactosidase activity was determined in situ by X-gal staining . The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species . The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells . The staining pattern was the same in either homologous or heterologous embryos . The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed . To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region . This gene fusion showed preferential expression in aboral ectoderm cells . However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid . These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.

J Bacteriol, 1990 Dec, 172(12), 7104 - 10
An expression vector for the archaebacterium Haloferax volcanii; Nieuwlandt DT et al.; The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology . We describe a shuttle-expression vector that can be selected for and maintained in either H . volcanii or Escherichia coli and permits the expression of cloned genes in H . volcanii . The vector, pWL204, was constructed by incorporating an H . volcanii tRNA(Lys) gene promoter into a derivative of the H . volcanii-E . coli shuttle vector pWL102 . The vector has been used to express a modified, intron-containing, H . mediterranei tRNA(Trp) gene (tRNA(Trp)-O167) . Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167 . Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.

Infect Immun, 1990 Dec, 58(12), 3909 - 13
Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis; Toye B et al.; A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV) . The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits . Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays . Antisera recognized MOMP from all 12 tested serovars of C . trachomatis but not from Chlamydia psittaci . In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I . High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies . Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited . Antisera were able to neutralize only those C . trachomatis serovars that bound antibodies in the dot blot assay . These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C . trachomatis serovars.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9454 - 8
Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions; Hsu MY et al.; The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome . The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA . This suggests that the 34-kilobase element was probably integrated into the E . coli genome by a mechanism related to transposition or phage integration . In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E . coli Dam methylase . Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.

J Immunol, 1990 Dec 1, 145(11), 3747 - 54
Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin; Borth W et al.; Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein . The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta . Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+) . Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect . Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time . Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta . In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups . Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+ . Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M . Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE . Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta . Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M . The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent . These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

J Virol, 1990 Dec, 64(12), 5757 - 63
Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA; Oertle S et al.; Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles . To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus . A point mutation in the protease (PR) active site completely prevents Pr76gag processing . The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it . A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA . This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging . These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.

Agric Biol Chem, 1990 Dec, 54(12), 3241 - 50
Extracellular production of human tumor necrosis factor-alpha by Escherichia coli using a chemically-synthesized gene; Nakamura S et al.; A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells . Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8 . The TNF mutant with two N-terminal amino acids deleted (N delta 2-TNF) was efficiently excreted into the culture medium by E . coli carrying the plasmid pEXTNF3 . In this clone, the signal peptide was correctly processed during the excretion . The E . coli-excreted N delta 2-TNF had higher antitumor activity than wild-type TNF or N delta 2-TNF produced intracellularly by E . coli.

J Steroid Biochem Mol Biol, 1990 Nov 30, 37(4), 481 - 90
The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90; Ohara-Nemoto Y et al.; The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter . The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM) . Glycerol gradient analysis of the E . coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins . However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM) . Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor . Radiosequence analysis of the recombinant steroid-binding domain expressed in E . coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly . However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.

Gene, 1990 Nov 30, 96(1), 29 - 36
Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA; Oden KL et al.; A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623) . Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated . It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.

Gene, 1990 Nov 30, 96(1), 23 - 8
High efficiency transformation of Escherichia coli with plasmids; Inoue H et al.; We have re-evaluated the conditions for preparing competent Escherichia coli cells and established a simple and efficient method (SEM) for plasmid transfection . Cells (DH5, JM109 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 10(9) cfu/microgram of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence . Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation . These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.

Gene, 1990 Nov 30, 96(1), 141 - 5
HU-1 mutants of Escherichia coli deficient in DNA binding; Goshima N et al.; We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors . The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively . HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities . DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA . These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.

Cell, 1990 Nov 30, 63(5), 1085 - 97
Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation; Clos J et al.; We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila . The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization . A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats . Drosophila HSF produced in E . coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro . In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction . These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

Science, 1990 Nov 30, 250(4985), 1259 - 62
Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence; Rauscher FJ 3rd et al.; The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein . However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated . A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli . The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides . The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli . A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity . These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.

Gene, 1990 Nov 30, 96(1), 17 - 22
A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, lambda pGY97; Vincze E et al.; It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging . Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA . This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity . Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain . These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E . coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability . The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation . This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.

Cell, 1990 Nov 30, 63(5), 1013 - 24
Cyclin activation of p34cdc2; Solomon MJ et al.; The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis . Activation is delayed even after the accumulation of cyclin to a critical threshold concentration . We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs . Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin . The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase . Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated . The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation.

Biochem Biophys Res Commun, 1990 Nov 30, 173(1), 141 - 8
Molecular cloning of mouse acid beta-galactosidase cDNA: sequence, expression of catalytic activity and comparison with the human enzyme; Nanba E et al.; A full-length cDNA coding for mouse lysosomal acid beta-galactosidase has been isolated on the basis of homology with the human gene . Catalytic activity toward 4-methylumbelliferyl beta-D-galactoside in the COS-1 cell expression system provided positive proof for its authenticity . The sequence analysis showed that the degree of similarity between the human and mouse enzymes was approximately 70% in the nucleotide sequence and nearly 80% in the amino acid sequence . The deduced primary amino acid sequences of the enzymes from the two species indicated that, of the seven possible N-glycosylation sites in the human enzyme, five are conserved in the mouse enzyme . Three additional possible N-glycosylation sites, not present in the human enzyme, are found in the primary amino acid sequence of the mouse enzyme . All seven cysteine residues in the mouse enzyme are conserved in the human enzyme . Although the nucleotide sequence could be aligned to 60% identity with the E . coli beta-galactosidase, similarity in the amino acid sequence was minimal.

Biochim Biophys Acta, 1990 Nov 30, 1087(3), 336 - 8
Nucleotide sequence of mouse HSP60 (chaperonin, GroEL homolog) cDNA; Venner TJ et al.; The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined . The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements.

Cell, 1990 Nov 30, 63(5), 941 - 8
Guanylyl cyclase is a heat-stable enterotoxin receptor; Schulz S et al.; Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors . We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin . GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains . Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity . In addition, heat-stable enterotoxin from E . coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C . The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors . These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.

Biochem Biophys Res Commun, 1990 Nov 30, 173(1), 231 - 9
Expression of DNA sequences containing neuron specific enolase gene in Escherichia coli; Dutta SK et al.; There is evidence that the gene for gamma-gamma enolase (neuron specific enolase, NSE) is regulated during cell differentiation and development, conserved in a variety of organisms and contains mRNA destabilizing sequences . In order to investigate further the mechanisms of these processes and to obtain large quantity of this protein, the NSE gene was isolated from neuroblastoma cells and cloned in E . coli using standard molecular biology techniques . The NSE gene expression was studied and the expressed protein (recombinant NSE) was characterized extensively . The recombinant NSE behaves like parental NSE in antisera specificity, resistance for chaotropic agents like urea, thermal stability at higher temperatures etc . The physical parameters like secondary structure, hydrophilicity, antigenic index and flexibility of the expressed protein were studied . The results of the present investigation collectively form the basis for initial investigations of how the expression of NSE gene is regulated . This is the first report where the recombinant NSE gene has been characterized so extensively.

J Chromatogr, 1990 Nov 28, 522, 171 - 7
Use of metal chelate affinity chromatography for removal of zinc ions from alkaline phosphatase from Escherichia coli; Lubinska VK et al.; Alkaline phosphatase from Escherichia coli (APEC) is not retained at 4 degrees C on a metal-free tris(carboxymethyl)ethylenediamine (TED) column, but at 15 degrees C the metalloenzyme becomes bound to the gel . Chromatography of phosphatase on metal-free TED gel indicates a decline in its enzymic activity and zinc content to about 26% and 40%, respectively . The activity of chromatographed APEC can be partially restored by addition of zinc ions, indicating that metal-free TED gel is capable of removing zinc ions from alkaline phosphatase.

J Chromatogr, 1990 Nov 28, 522, 163 - 70
Reversed-phase chromatography of Escherichia coli ribosomal proteins . Correlation of retention time with chain length and hydrophobicity; Champney WS; The elution times of 52 bacterial ribosomal proteins from a C4 reversed-phase column have been predicted . The prediction is based upon the use of the hydrophobicity coefficients for the protein amino acid content as defined by Guo et al . {J . Chromatogr., 359 (1986) 499-518} . A strong positive correlation was observed when the difference between predicted and observed protein retention time was plotted against the product of net hydrophobicity and natural log of protein chain length . Observations with this class of related proteins strengthens and extends the observations of Mant et al . {J . Chromatogr., 476 (1989) 363-375} . Observed deviations from predicted chromatographic behavior can be explained for several proteins which elute as dimers or which have modified amino acid residues.

Biochemistry, 1990 Nov 27, 29(47), 10590 - 3
Remodeling hexose-1-phosphate uridylyltransferase: mechanism-inspired mutation into a new enzyme, UDP-hexose synthase; Kim J et al.; Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166 . To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted . To determine whether the function of the deleted imidazole in these mutants could be carried out by the imidazole ring in uridine 5'-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole . The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates . The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose . Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction . The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase . The value of kcat in the direction of UDP-glucose formation is 1.31 +/- 0.01 s-1, compared with 350 s-1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 +/- 0.4 s-1, compared with 960 s-1 for the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Nov 27, 29(47), 10659 - 63
The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit; Lolkema JS et al.; The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation . Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate . At equilibrium, bound mannitol is situated at the periplasmic side of the membrane . The apparent binding constant is 40 nM for the intact membranes . Solubilization of the membranes in detergent decreases the affinity by about a factor of 2 . Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities . These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.

Biochemistry, 1990 Nov 27, 29(47), 10695 - 702
Sequence requirements of the hammerhead RNA self-cleavage reaction; Ruffner DE et al.; A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study . The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined . Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage . In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex . In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure . All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA . The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

FEBS Lett, 1990 Nov 26, 275(1-2), 91 - 4
Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/beta-galactosidase/galactokinase; Ljungcrantz P et al.; The in-frame gene fusion between 3 enzymes, galactose dehydrogenase, beta-galactosidase and galactokinase, is described . The purified artificial tripartite enzyme displayed all three enzymic activities . Two major forms of the hybrid protein were found, consisting of 4 and 8 subunits respectively, but other forms could also be identified . Each subunit was made up of one monomer each of galactose dehydrogenase, beta-galactosidase and galactokinase . Proximity effects exhibited by the hybrid enzyme could be demonstrated using {14C}galactose as a reporter molecule.

Eur J Biochem, 1990 Nov 26, 194(1), 103 - 8
Independent folding of individual components in hybrid proteins . Evidence that the carboxy-terminal 135 residues of the LexA repressor constitute a single autonomous domain; Slilaty SN et al.; Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84-Gly85 peptide bond near the center of the molecule . The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein . The entire self-cleavage activity of LexA has been found to lie within a 135-residue tryptic fragment extending from Leu68 to the end of the protein at Leu202 . Since the activity of self-cleavage is dependent on the proper three-dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135-residue carboxy-terminal domain of LexA by applying a protein fusion approach . A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self-cleavage activity has been retained . The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used . Loss of self-cleavage was found to be a function of amino- or carboxy-terminal deletions in the self-cleaving LexA component of the fusion proteins . The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6641 - 7
Statistical analysis of nucleotide sequences; Stuckle EE et al.; In order to scan nucleic acid databases for potentially relevant but as yet unknown signals, we have developed an improved statistical model for pattern analysis of nucleic acid sequences by modifying previous methods based on Markov chains . We demonstrate the importance of selecting the appropriate parameters in order for the method to function at all . The model allows the simultaneous analysis of several short sequences with unequal base frequencies and Markov order k not equal to 0 as is usually the case in databases . As a test of these modifications, we show that in E . coli sequences there is a bias against palindromic hexamers which correspond to known restriction enzyme recognition sites.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6537 - 44
Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2; Tate W et al.; An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site . Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor . An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition . An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination . The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified . RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found . If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted . These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6517 - 22
Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement; Donly BC et al.; The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation . We have constructed fusions between the genes for RF-2 and E . coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable . The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site . The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release) . These are the first examples of release factor mutants selectively active for only one of these function . In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis . This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.

J Biol Chem, 1990 Nov 25, 265(33), 20673 - 7
Cellular processing of the interleukin-2 fusion toxin DAB486-IL-2 and efficient delivery of diphtheria fragment A to the cytosol of target cells requires Arg194; Williams DP et al.; We have used site-directed mutagenesis to examine the role played by Arg191, Arg193, and Arg194 of the fusion toxin DAB486-IL-2 in the intoxication of high affinity interleukin-2 receptor-bearing T-lymphocytes . These arginine residues are positioned in the proteolytically sensitive 14-amino acid loop subtended by the disulfide bond between Cys187 and Cys202 in this fusion toxin . DAB486-IL-2 was formed by the genetic substitution of the native diphtheria toxin receptor binding domain with human interleukin-2 (Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B., and Murphy, J.R . (1987) Protein Eng . 1, 493-498) . We demonstrate that substitution of Arg194 with Gly results in a 1000-fold loss of DAB486-IL-2 potency . Since trypsin "nicking" of the Gly194 mutant restores biologic activity, we conclude that Arg194 is required for the cellular processing of the fusion toxin which results in the release of fragment A into the cytosol.

J Biol Chem, 1990 Nov 25, 265(33), 20609 - 15
Expression of wild-type and mutated forms of the catalytic (alpha) subunit of Caenorhabditis elegans casein kinase II in Escherichia coli; Hu E et al.; A full-length Caenorhabditis elegans cDNA that encodes the alpha subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCK alpha . Escherichia coli DE21 lysozyme S that was transformed with pCK alpha expressed soluble, catalytically active casein kinase II alpha upon induction with isopropyl beta-D-thiogalactopyranoside . The expressed alpha subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose . The Mr values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 42,000) or calculated from hydrodynamic measurements (s20,w = 3.3 S, Stokes radius = 2.8 nm, Mr = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric . The native holoenzyme and the expressed alpha subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin . However, the kcat for E . coli-derived alpha was only 9% of the kcat for the native holoenzyme, and catalytic activity was not stimulated by polyamines . Recombinant casein kinase II alpha aggregates at low ionic strength, and the aggregation is partially reversible . A mutant alpha subunit in which Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis . The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g . IC50 = 0.3 micrograms/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased approximately 70-fold . Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain . The successful expression of casein kinase II alpha in E . coli will facilitate the analysis of the structural basis for functional domains in this enzyme.

J Biol Chem, 1990 Nov 25, 265(33), 20421 - 9
Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase; Marceau M et al.; We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase . These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites . The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid) . The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides . C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively . The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity . The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes) . C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase . Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A . These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.

J Biol Chem, 1990 Nov 25, 265(33), 20085 - 6
Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis; Lerner CG et al.; In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease . The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene . The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding . Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3% . From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified . The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.

J Biol Chem, 1990 Nov 25, 265(33), 20069 - 72
Minimum substrate sequence for signal peptidase I of Escherichia coli; Dev IK et al.; The minimum substrate sequence recognized by signal peptidase I (SPase I or leader peptidase) was defined by measuring the kinetic parameters for a set of chemically synthesized peptides corresponding to the cleavage site of the precursor maltose binding protein (pro-MBP) . The minimum sequence of a substrate hydrolyzed by SPase I at a measurable rate was the pentapeptide Ala-Leu-Ala decreases Lys-Ile . The rates of hydrolysis of this substrate, however, were several hundred-fold lower than those observed for the maturation of MBP in Escherichia coli, suggesting that in addition to these minimal sites involved in recognition, other features of pro-MBP are also needed for the optimal rate of signal peptide cleavage by SPase I . One parameter may be the length of the polypeptide chain . Studies of the synthetic peptides showed that decreasing the length of the polypeptide chain of substrates decreased the substrate efficiency measured as kcat/Km . However, in one case a decrease in the length of a peptide corresponding to -7 to +3 positions of pro-MBP to a nonapeptide (-7 to +2) increased the substrate efficiency by about 900-fold . The nonapeptide is the most efficient substrate for the enzyme in vitro so far reported . It is speculated that better peptide substrates are the ones which are able to adopt folded structures.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6659 - 63
Purification and characterization of a DNA polymerase from the cyanobacterium Anacystis nidulans R2; Lin HJ et al.; A DNA polymerase has been highly purified from Anacystis nidulans R2 . Electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels revealed that the final fraction contains three bands of Mr 107,000, 93,000, and 51,000, respectively . Analysis of purified DNA polymerase activity in situ indicates that of the three polypeptides the Mr 107,000 species has the catalytic activities . The native molecular weight of the enzyme was estimated by glycerol gradient sedimentation to be 100,000 . The enzyme has an absolute requirement for a divalent cation . Mg2+ can be replaced with Mn2+, but the DNA polymerase is less active . Potassium chloride stimulates the enzyme, while potassium phosphate has no apparent effect . The enzyme is active over a pH range from 7.5 to 9.5 in 50mM Tris-HCl buffer . The ability of the cyanobacterial DNA polymerase to use activated DNA as a template, its associated 3'----5' and 5'----3' exonuclease activities, as well as its resistance to N-ethylmaleimide, dideoxynucleotides, arabinosyl-CTP and aphidicolin suggest a similarity between this enzyme and E . coli DNA polymerase I . This is the first characterization of a DNA polymerase from a cyanobacterium.

J Biol Chem, 1990 Nov 25, 265(33), 20384 - 9
Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli . Bromopyruvate inactivation and labeling of glutamate 45; Vlahos CJ et al.; Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity . Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate . Inactivation studies with {14C} bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45 . Incubation of the enzyme with excess {14C}bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well . Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues . The results indicate that Glu-45 of E . coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed.

J Biol Chem, 1990 Nov 25, 265(33), 20293 - 301
Structure-function studies of the SERPIN plasminogen activator inhibitor type 1 . Analysis of chimeric strained loop mutants; Lawrence DA et al.; Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop . The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1 . Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin . Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1 . Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA . This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.

J Chromatogr, 1990 Nov 23, 521(2), 267 - 77
Purification of a recombinantly produced transmembrane protein (gp41) of HIV I; Soutschek E et al.; The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies . The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli . The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell . Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures . Growth conditions of the recombinant E . coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen . Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components . For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used . After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed . The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important . Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.

Nature, 1990 Nov 22, 348(6299), 339 - 42
(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL; Lissin NM et al.; The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone . It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates . As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.

Biochemistry, 1990 Nov 20, 29(46), 10455 - 60
Formation and stability of repairable pyrimidine photohydrates in DNA; Boorstein RJ et al.; Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases {Boorstein et al . (1989) Biochemistry 28, 6164-6170} . Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU) . When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil) . To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation . Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h . Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C . Uracil hydrate and uracil were also formed in irradiated poly(dG-dC) . These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil . The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.

J Mol Biol, 1990 Nov 20, 216(2), 411 - 24
Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP; Brunie S et al.; The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms) . In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms . In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A . The position of the zinc ion has been confirmed and is located near the active site . The tryptic fragment is composed of two globular domains . The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted . The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site . The second domain, up to Pro526, is mainly alpha-helical . The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains . The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located . The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme . The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively . The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose . One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP . The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule . The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1990 Nov 20, 216(2), 375 - 84
Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase . Role of the C-terminal region of the regulatory chains; Xi XG et al.; In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig . 1) . In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis . The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites . The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites . This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

J Mol Biol, 1990 Nov 20, 216(2), 353 - 62
Structural study of the yeast RNA polymerase A . Electron microscopy of lipid-bound molecules and two-dimensional crystals; Schultz P et al.; Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers . The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization . Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry . The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end . These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove . Additional structural features were observed and compared to the Escherichia coli enzyme.

J Mol Biol, 1990 Nov 20, 216(2), 299 - 310
Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA; Moine H et al.; Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one . The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site . The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm . This binding is restricted to the stem-loop structure of the second site . However, the interaction of the enzyme with this site fails to occlude ribosome binding . tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex . Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase . This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

J Mol Biol, 1990 Nov 20, 216(2), 289 - 98
FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site; Schwartz CJ et al.; The FLP recombinase of the 2 mu plasmid of Saccharomyces cerevisiae binds to a target containing three 13 base-pair symmetry elements called a, b and c . The symmetry elements b and c are in direct orientation while the a element is in inverted orientation with respect to b and c on the opposite side of an eight base-pair core region . Each symmetry element acts as a binding site for the FLP protein . The FLP protein can form three different complexes with the FLP recognition target (FRT site) according to the number of elements within the site that are occupied by the FLP protein . Binding of FLP to the FRT site induces DNA bending . We have measured the angles of bends caused by the binding of the FLP protein to full and partial FRT sites . We find that FLP induces three types of bend in the FRT-containing DNA . The type I bend is approximately 60 degrees and results from a molecule of FLP bound to one symmetry element . The type II bend is greater than 144 degrees and results from FLP molecules bound to symmetry elements a and b . The type III bend is approximately 65 degrees and results from FLP proteins bound to symmetry elements b and c . Certain FLP proteins that are defective in recombination can generate the type I and type III bends but are impaired in their ability to induce the type II bend . We discuss the role of bending in FLP-mediated recombination.

J Mol Biol, 1990 Nov 20, 216(2), 275 - 87
Specific sequences downstream from -6 are not essential for proper and efficient in vitro utilization of the Escherichia coli lactose promoter; Lorimer DD et al.; A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA . These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro . In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter . A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream . These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process . Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level . No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products . Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter . Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions . A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered . However, if a "P3" site does exist, it must lie between P1 and P2 . Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme . The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter.

J Mol Biol, 1990 Nov 20, 216(2), 261 - 73
Positive and negative regulation of transcription by a cleavage product of Ada protein; Akimaru H et al.; The 39,000 Mr Ada protein of Escherichia coli that carries two distinct methyltransferase activities and activity to promote transcription of the ada and the alkA genes is cleaved by a cellular proteinase . As a result, the 20,000 and the 19,000 Mr proteins are formed, which are derived from the N-terminal and the C-terminal halves of the protein, respectively . To elucidate the molecular mechanism of transcriptional control by Ada protein, the N-terminal 20,000 Mr protein was overproduced by manipulating the cloned ada gene . The protein possessed an activity to transfer a methyl group from the methylphosphotriester of the alkylated DNA to its own molecule and retained the potential to promote transcription of the alkA gene . The methylated form of the 20,000 Mr proteins binds to the proper alkA regulatory sequence, as does the intact Ada protein, and facilitates further binding of RNA polymerase to the promoter, thus forming an active transcription initiation complex . The non-methylated 20,000 Mr protein was incapable of binding itself or supporting RNA polymerase binding to the alkA promoter . When the 20,000 Mr protein was produced under the control of the lac promoter in E . coli and then exposed to a methylating agent, a considerable amount of 3-methyladenine-DNA glycosylase II, the product of the alkA gene, was formed . Thus, the results obtained in in vitro experiments were confirmed by the events observed in vivo . The methylated 20,000 Mr protein also binds to the ada promoter; however, it does not facilitate further binding of RNA polymerase to the promoter nor does it promote ada transcription in vitro . These findings indicate that the N-terminal half of Ada protein is mainly responsible for recognition of and binding to alkA and the ada regulatory sequence . The methylated 20,000 Mr protein occupies the same region of the ada promoter to which the intact Ada protein would bind, thereby suggesting that it acts as a repressor for expression of the ada gene . The ada transcription promoted by the Ada protein was greatly inhibited by the methylated, but not the non-methylated, form of the 20,000 Mr protein . In an in vivo system, formation of the 20,000 Mr protein leads to inhibition of transcription from the ada promoter . We suggest that termination of the adaptive response may come about by proteolytic cleavage of the Ada protein, the result being a loss of the activator as well as formation of the repressor for ada transcription.

J Mol Biol, 1990 Nov 20, 216(2), 207 - 11
Purification and secondary structure determination of simian immunodeficiency virus p27; Burns NR et al.; We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus . Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements . These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.

J Mol Biol, 1990 Nov 20, 216(2), 195 - 9
Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant; Huang XT et al.; An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225) . The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis . The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation . The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation . The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle . These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure . DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA.

J Mol Biol, 1990 Nov 20, 216(2), 335 - 52
Energetics of RecA-mediated recombination reactions . Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle; Rosselli W et al.; We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis . The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used . We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions . The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction . This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction.

J Mol Biol, 1990 Nov 20, 216(2), 189 - 94
Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons; Cohen J et al.; Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine . Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli . To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator . This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E . coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization . This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis . A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.

Cell, 1990 Nov 16, 63(4), 687 - 95
The hepatitis B virus-encoded transcriptional trans-activator hbx appears to be a novel protein serine/threonine kinase; Wu JY et al.; To study the functional mechanism of the hepatitis B virus (HBV) X (hbx) gene product, we have expressed the hbx protein in E . coli and purified it by HPLC . The purified hbx protein was shown to be active in transactivating transcription directed by the LTR sequence of HIV-1 . The hbx protein was found to have an intrinsic serine/threonine protein kinase activity . The hbx protein was detected in hepatitis B virions, and tryptic phosphopeptide maps of the hbx protein phosphorylated in the virion and of the in vitro phosphorylated bacterially expressed hbx protein were similar . Inactivation of the hbx protein by heat, protein-denaturing agents, or an ATP affinity analog, p-fluorosulfonylbenzoyl 5'-adenosine, resulted in loss of both protein kinase activity and trans-activation activity . These results suggest that the HBV-encoded trans-activator hbx is a novel protein kinase.

Cell, 1990 Nov 16, 63(4), 773 - 81
Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL; Kim S et al.; To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants . The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination . It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain . This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.

Cell, 1990 Nov 16, 63(4), 815 - 25
Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun; Baichwal VR et al.; Analysis of transcriptional activation properties of c-Jun chimeras in different cell lines suggests that it contains an activator domain (A1) that is negatively regulated by a cell type-specific inhibitor . A regulatory domain of c-Jun, delta, previously identified by in vitro experiments, also regulates transcriptional activation by c-Jun in vivo . The delta domain facilitates or stabilizes the interaction of the cellular inhibitor with A1 . v-Jun, which lacks delta, is a stronger transcriptional activator than c-Jun, since its activity is not efficiently repressed by the cellular inhibitor . In vitro transcription with chimeric Jun proteins and extracts from different cell types confirms that the A1 and delta domains are repressed in a cell type-specific manner . These findings implicate a specific cellular factor in the negative regulation of c-Jun activity and suggest a molecular basis for the observed difference in transcriptional properties between v-Jun and c-Jun.

J Biol Chem, 1990 Nov 5, 265(31), 19091 - 9
Characterization of transcriptional initiation from promoters P1 and P2 of the pyrBI operon of Escherichia coli K12; Donahue JP et al.; Expression of the pyrBI operon of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by pyrimidine availability, primarily through an attenuation control mechanism, and also appears to be subject to stringent control . Previous in vitro transcription studies indicated that the pyrBI operon is transcribed from tandem promoters designated P1 and P2, which appeared to be of similar strength . In this study, we characterized these promoters in detail and examined their role in pyrBI expression . Our results show that although transcription is initiated at both promoters in vivo, greater than 99% of the pyrBI transcripts are initiated at promoter P2, indicating that this is the only physiologically significant promoter . The level of transcripts initiated at promoter P2 was found to be higher in cells grown under pyrimidine-limiting conditions compared to that in cells grown under conditions of pyrimidine excess, indicating pyrimidine-mediated regulation at the level of transcriptional initiation . In vitro characterization of transcription from the pyrBI promoter region showed that non-physiological reaction conditions used in the original identification of the two promoters greatly overestimated the strength of promoter P1 . Further in vitro characterization of the two promoters showed that transcription from promoter P2, but not from promoter P1, is inhibited by guanosine tetraphosphate and exhibits salt and heparin sensitivity typical of a stringently controlled promoter . In addition, heparin-challenge experiments revealed a UTP-induced instability of transcriptional initiation complexes at promoter P2 which may be of regulatory significance.

J Biol Chem, 1990 Nov 15, 265(32), 19990 - 5
The tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product; Kanei-Ishii C et al.; In the DNA-binding domain of the c-myb protooncogene product (c-Myb) which consists of three repeats of 51-52 amino acids, there are 3 perfectly conserved tryptophans in each repeat . Site-directed mutagenesis of these tryptophans showed that any single or multiple mutations of tryptophan to hydrophilic residues or alanine abolished or greatly reduced the sequence-specific DNA-binding activity, but mutations to hydrophobic amino acids retained considerable activity . Raman spectroscopic study showed that these tryptophans were buried in the protein core . These 3 tryptophans are proposed to form a cluster in the hydrophobic core in each repeat . This hypothetical structure is referred to as the "tryptophan cluster," and it may represent a characteristic property of a group of DNA-binding proteins including the myb- and ets-related proteins.

J Biol Chem, 1990 Nov 15, 265(32), 19582 - 7
The absence of a m7G cap on beta-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation; Fletcher L et al.; beta-Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m7G cap structure (m7G(5')ppp(5')N) . The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ . In the S30 system the uncapped forms of beta-globin mRNA and AMV RNA 4 are much less active (greater than or equal to 10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active . The low activity of uncapped beta-globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system . Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of beta-globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation . For beta-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM) . The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of beta-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5- to 2-fold) than the concentrations required for the capped forms . With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same . The translation of the capped and uncapped forms of beta-globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m7GTP in the partially purified system containing low concentrations of eIF-4F . Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP . These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs.

J Biol Chem, 1990 Nov 15, 265(32), 19560 - 7
Electron transfer from menaquinol to fumarate . Fumarate reductase anchor polypeptide mutants of Escherichia coli; Westenberg DJ et al.; Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant . The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone . To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes . Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized . The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices . Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions . All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate . Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees . The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.

J Biol Chem, 1990 Nov 15, 265(32), 19551 - 9
Peptide and protein carboxyl-terminal labeling through carboxypeptidase Y-catalyzed transpeptidation; Berne PF et al.; A survey of carboxypeptidase Y-catalyzed carboxyl-terminal modification of short peptides in the presence of various amino acids revealed that transpeptidation occurred in significant yield only with peptides containing a proline at the penultimate or antepenultimate position . For these peptides, transpeptidation was shown to occur specifically at the carboxyl side of the proline, thus suggesting a determining role of this residue for transpeptidation . Two model peptides, YPFP-GPI and YPFVEPI, were studied in detail . Initial yields of transpeptidation in the presence of various nucleophiles were compared . Among natural amino acids, the highest yield was obtained with methionine, followed by other amino acids bearing hydrophobic side chains . In order to transpose the method of transpeptidation to a protein, a variant of Escherichia coli methionyl-tRNA synthetase bearing the carboxyl-terminal Glu-Pro-Met sequence was genetically created . Under the conditions optimized for the transpeptidation of YPF-VEPI with methionine, this protein could be labeled specifically at its carboxyl-terminal end . Moreover, the parameters of the labeling reaction were in agreement with those observed in the transpeptidation of the model peptide.

J Biol Chem, 1990 Nov 15, 265(32), 19397 - 400
Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22; Keiper BD et al.; We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22 . A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes . In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes . Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.

J Biol Chem, 1990 Nov 15, 265(32), 19377 - 80
Molecular cloning, sequencing, and expression of mouse ferrochelatase; Taketani S et al.; The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody . Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained . These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions . From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692) . The cDNA allows for the expression of active ferrochelatase by transfected culture cells . RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites . Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide . The band pattern of the RNA of the mouse liver was the same as that of the MEL cells . Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.

Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1230 - 5
Molecular cloning and expression of human arachidonate 12-lipoxygenase; Yoshimoto T et al.; The cDNA for a 12-lipoxygenase was isolated from cDNA library of human erythroleukemia cells . The cDNA had an open reading frame encoding 663 amino acids with a calculated molecular weight of 75,513 . The deduced amino acid sequence of human 12-lipoxygenase exhibited 41.5%, 65.3% and 65.4% identity with human 5-lipoxygenase, human 15-lipoxygenase and porcine 12-lipoxygenase, respectively . Blot hybridization analysis of RNA from human erythroleukemia cells demonstrated a single species (3.1 kb) of mRNA with the cDNA probe for 12-lipoxygenase of these cells, but not with the cDNA for porcine leukocyte enzyme . The cytosol of Escherichia coli transformed with a recombinant pUC19 plasmid oxygenated the position 12 of arachidonic acid.

Arch Biochem Biophys, 1990 Nov 15, 283(1), 96 - 101
The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli; Angeles TS et al.; The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined . Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+ . Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor . Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme . The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism . Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.

Arch Biochem Biophys, 1990 Nov 15, 283(1), 141 - 9
Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli; Boutelje J et al.; Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions . The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically . To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli . In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A . The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease . A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond . Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein . The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus . The fusion protein also provided a source of authentic protease . The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.

Biochem J, 1990 Nov 15, 272(1), 107 - 12
Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4) . InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx . 50,000 on SDS/polyacrylamide gels . Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000 . Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive . A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library . The 5' end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase . The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II . When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity . Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

J Biol Chem, 1990 Nov 15, 265(32), 19679 - 84
Molecular structure of microtubule-associated protein 2b and 2c from rat brain; Kindler S et al.; Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced . The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli . The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain . The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2 . This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence . Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively . A third homologous domain of unknown function lies in a central region of MAP2b . Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2 . Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.

J Biol Chem, 1990 Nov 15, 265(32), 19502 - 6
Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides; Cadd GG et al.; Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties . We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits . The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined . Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes . However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha . Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation . Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile) . These sequences form part of a pseudosubstrate site thought to interact with the C subunit . Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides . Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30% . These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1157 - 62
Protein conformational "annealing": binding to cytochrome c refolds the active site region of recombinant cytochrome c peroxidase; Hake R et al.; When initially isolated with heme reconstitution, recombinant cytochrome c peroxidase molecules exhibit a conformation, revealed by visible spectra which observably differ from the corresponding holo proteins isolated from yeast . Binding yeast iso-1 cytochrome c to these recombinant cytochrome c peroxidases (either in solution or via an affinity column) catalyses a local refolding of the recombinant proteins to a form that is indistinguishable from the native (yeast) protein.

Biochem J, 1990 Nov 15, 272(1), 223 - 9
Annexin proteins PP4 and PP4-X . Comparative characterization of biological activities of placental and recombinant proteins; Romisch J et al.; The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield . The proteins were purified to homogeneity . The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts . Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria . Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test.

Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1217 - 23
Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichia coli; Shinkai A et al.; SecA is an essential component of the protein secretory machinery of Escherichia coli . SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis . The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures . It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation . SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution . Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol . It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.

J Biol Chem, 1990 Nov 15, 265(32), 19848 - 52
Nucleosome linking number change controlled by acetylation of histones H3 and H4; Norton VG et al.; High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V . G., Imai, B . S., Yau, P., and Bradbury, E . M . (1989) Cell 57, 449-457) . Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B . This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4) . Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle.

Gene, 1990 Nov 15, 95(2), 223 - 30
Stable, high-level expression of a carcinoembryonic antigen-encoding cDNA after transfection and amplification with the dominant and selectable asparagine synthetase marker; Cartier M et al.; The introduction and expression of cloned genes in a wide variety of animal cells requires the convenient use of dominant selectable markers . Very few of these markers can be amplified in copy number, a necessary feature if variable and high-level expression of the gene of interest is required . We describe the successful dominant transfection and amplification of a vector containing the Escherichia coli asparagine synthetase (AS)-encoding gene, asnA, transfected into a variety of human and rodent cell lines . An unlinked co-transfected expression vector containing the CEA cDNA, encoding human carcinoembryonic antigen, can be co-amplified with the asnA marker leading to extremely high levels of CEA synthesis . In addition, we show that the expression of both the asnA marker and the co-transfected CEA construct are stable in normal and amplified transfectants after prolonged culture in the absence of selective pressure.

Anal Biochem, 1990 Nov 15, 191(1), 65 - 9
Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support; Vener AV et al.; A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli . Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol . In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding . The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase . Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity.

Anal Biochem, 1990 Nov 15, 191(1), 35 - 40
The use of ubiquitin-peptide extensions as protein kinase substrates; Yoo Y et al.; Four ubiquitin-peptide extensions prepared as cloned products in E . coli were tested as casein kinase II substrates . Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II . The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions . Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs . Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites . Such ubiquitin extension proteins should prove valuable as protein kinase substrates.

Eur J Biochem, 1990 Nov 13, 193(3), 801 - 6
The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin; Banyai L et al.; A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109 . The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site . Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding . In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.

Eur J Biochem, 1990 Nov 13, 193(3), 643 - 50
Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol; Rheinberger HJ et al.; Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively . Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+ . Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2 . The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well . This observation resolves a current controversy concerning the active fraction of ribosomes . The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine) . Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA . The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).

Biochemistry, 1990 Nov 13, 29(45), 10387 - 93
Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase; Aggeler R et al.; Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme . All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II) . These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex . Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment . ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf . an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment . The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit . mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments . The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme . The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.

Eur J Biochem, 1990 Nov 13, 193(3), 817 - 25
Purification and characterization of acyl carrier protein from two cyanobacteria species; Froehlich JE et al.; The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184) . Both ACPs have been purified to homogeneity utilizing a three-column procedure . Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A . variabilis ACP was purified 1040-fold with 50% yield . Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A . variabilis were achieved . Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs . Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region . Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E . coli ACP than spinach ACP I . Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody . SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A . variabilis ACP showed that cyanobacteria ACPs co-migrated with E . coli ACP and had relative molecular masses of 18,100 and 17,900 respectively . Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase . A . variabilis and Synechocystis 6803 ACP could be acylated using E . coli acyl ACP synthetase.

FEBS Lett, 1990 Nov 12, 274(1-2), 85 - 8
In vivo evidence for FhuA outer membrane receptor interaction with the TonB inner membrane protein of Escherichia coli; Gunter K et al.; FhuA outer membrane receptor activity of Escherichia coli K-12 depends on the TonB inner membrane protein . The naturally occurring degradation of the TonB protein could be prevented by the FhuA receptor protein . Mutated TonB proteins could only be stabilized by mutated FhuA proteins when they functionally interacted in the uptake of ferrichrome across the outer membrane.

FEBS Lett, 1990 Nov 12, 274(1-2), 199 - 202
Carbachol mimics phorbol esters in its ability to enhance cyclic GMP production by STa, the heat-stable toxin of Escherichia coli; Crane JK et al.; STa, the heat-stable enterotoxin of Escherichia coli, stimulates membrane-bound guanylate cyclase in enterocytes, elevates cyclic GMP, and results in intestinal secretion of ions and fluid . Using the T84 colon carcinoma cell line as a model . Weikel et al . reported that phorbol esters enhance STa-stimulated cyclic GMP production by 60-140% {(1990) Infect . Immun . 58, 1402-1407} . In the present report we demonstrate that the acetylcholine analog carbachol enhanced toxin-stimulated cyclic GMP accumulation in intact T84 cells by 50-100% and that this effect was blocked by 10 microM atropine and 10 microM sphingosine . Pertussis toxin treatment of the T84 cells did not affect the subsequent response to carbachol . Carbachol, which elevates intracellular calcium in these cells, may act through protein kinase C to enhance cyclic GMP production.

FEBS Lett, 1990 Nov 12, 274(1-2), 9 - 11
The plant mitochondrial F1-ATPase . The identity of the delta' (20 kDa) subunit; Horak A et al.; The N-terminal amino acid sequence of the 20 kDa (delta') subunit of the turnip (Brassica napus L.) mitochondrial F1-ATPase has been determined . Comparison of the sequence obtained with those of the epsilon subunits of chloroplast CF1, E . coli F1 and the delta subunit of bovine F1 shows that the turnip delta' subunit is another member of this family of homologous proteins . The delta' subunit of sweet potato F1-ATPase {(1989) J . Biol . Chem . 264, 3183-3186} is very similar to the turnip sequence and thus can also be considered to belong to this family.

Mol Microbiol, 1990 Nov, 4(11), 2003 - 6
Corrected sequence of the mannitol (mtl) operon in Escherichia coli; Jiang W et al.; The previously published sequences of the operator-promoter region of the mannitol operon of Escherichia coli and of the mtlD gene have been found to contain a number of errors . The major conclusions reported previously were correct, but additionally it is now clear that a C-terminal portion of mannitol-1-phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino-terminal region of human liver fructose-6-phosphate-2-kinase:fructose-2,6-bisphosphatase.

J Ind Microbiol, 1990 Nov, 6(3), 199 - 206
Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins in Escherichia coli; Keller J et al.; The major leftward early promoter of phage lambda pL, has frequently been used to drive expression of heterologous genes in Escherichia coli . pL is typically maintained fully repressed by the lambda cl protein . When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences . One method of derepressing pL involves exposing cells to nalidixic acid, which results in the "activation" of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl . Activated RecA also mediates the cleavage of the E . coli LexA protein, resulting in induction of the SOS regulon (at least 15 E . coli genes, including recA) . We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions . One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind-) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage . These mutations were introduced into a strain carrying a cl+ defective lysogen . Synthesis of two heterologous proteins, human alpha 1-antitrypsin and a fusion protein partially derived from the Plasmodium falciparum circumsporozoite surface antigen, was examined in the wild-type and mutant strains . The maximum alpha-1 antitrypsin concentration achieved was improved by 50% when the recA o strain was used rather than the wild-type; however, only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein were observed . Use of the lexAind- strain resulted in a decrease in the maximum concentration attained for both heterologous products.

Nucleic Acids Res, 1990 Nov 11, 18(21), 6331 - 8
On the movement and alignment of DNA during 120 degrees pulsed-field gel electrophoresis; Whitcomb RW et al.; The displacement per pulse of lambda, T4, and G DNA during pulsed-field agarose gel electrophoresis has been measured for a fine mesh of pulse durations T between 0.02 and 120 s . The slopes of these curves show that the DNA moves by two distinct processes, designated 1 and 2, depending upon the pulse duration T . Process 1 operates at short T and causes dx/dT to decrease gradually with increasing T . This process is independent of molecular weight M . Process 2 is effective at longer T and causes dx/dT to rise sharply in sigmoidal fashion at a value of T which increases as M1.2, finally reaching a plateau of 1.4 microns/s for E = 4 V/cm . The shape of the dx/dT curve and its dependence on M lead directly to 4 zones of separation in plots of mobility vs M for different T . The alignment of the 3 DNAs during PFGE was measured by fluorescence-detected linear dichroism for E between 4 and 10 V/cm . These results are used in developing a molecular understanding of the mobility data.

Nucleic Acids Res, 1990 Nov 11, 18(21), 6215 - 22
Gene F of plasmid RSF1010 codes for a low-molecular-weight repressor protein that autoregulates expression of the repAC operon; Maeser S et al.; The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4 . We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene . The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4 . To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro . Purification was based on a gel retardation assay for detection of P4-specific DNA binding . Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions . The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.

Nucleic Acids Res, 1990 Nov 11, 18(21), 6325 - 30
Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity; Ushida C et al.; A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region . The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides . DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site . The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids . Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription . In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer . We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action . The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.

Nucleic Acids Res, 1990 Nov 11, 18(21), 6339 - 45
Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes; Brown CM et al.; An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported . In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families . Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes . For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes . The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions . We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal . A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.

Vet Rec, 1990 Nov 10, 127(19), 475 - 7
Use of scanning electron microscopy to investigate dental calculus in dogs; Robert JC et al.; The formation of dental calculus in dogs is a major problem . Scanning electron microscopy of tartar specimens from dogs revealed on the outer surface of the plaque polymorphic configurations, more or less arranged as free filaments, corn-cobs or swab-like structures . Uninhabited bacterial recesses were found on the inner surface of the calculus . Calcification may occur between or within the bacteria . Elucidating the mechanisms of calculus formation should help in the development of prophylactic measures.

Biochemistry, 1990 Nov 6, 29(44), 10255 - 61
Chorismate mutase-prephenate dehydrogenase from Escherichia coli . 2 . Evidence for two different active sites; Turnbull J et al.; The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated . The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites . Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction . (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction . However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions . (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction . The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.

Biochemistry, 1990 Nov 6, 29(44), 10245 - 54
Chorismate mutase-prephenate dehydrogenase from Escherichia coli . 1 . Kinetic characterization of the dehydrogenase reaction by use of alternative substrates; Turnbull J et al.; The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP . Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates . The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically . As judged by their V/K values, all analogues were poorer substrates than prephenate . The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate . Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain . With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation . By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible . Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction . The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials . An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.

Eur J Pharmacol, 1990 Nov 6, 190(1-2), 185 - 92
Endotoxin-induced impairment of vascular smooth muscle contractions elicited by different mechanisms; Biguad M et al.; The current study was designed to analyse the mechanisms which are impaired in the vascular hyporeactivity to contractile agents induced by E . coli lipopolysaccharide endotoxin (LPS) . Endothelium-denuded aortic rings were prepared from thoracic aorta removed from control and LPS-pretreated rats (20 mg/kg i.p., 4 h before the experiment) . In order to determine whether LPS treatment altered the contractile components that depend on intracellular calcium release and extracellular calcium entry to the same extent, rings were contracted under various experimental conditions . The responses elicited by indanidine, phenylephrine (without and with nitrendipine 1 microM), (-) Bay K 8644, (+) S 202-791 and the calcium ionophore calimycin in the presence of 1.25 mM external CaCl2 were all impaired by LPS pretreatment (maximal contractions 19, 63, 44, 28, 22 and 22% of controls, respectively) . Concentration-effect curves for CaCl2 made in depolarizing medium (KCl 40 and 100 mM) and in the presence of calimycin (3 microM) were shifted to the right in rings from LPS-pretreated rats . However, the LPS-induced depression of contraction was overcome by the addition of CaCl2 (up to 30 mM) . Additionally, in the absence of external CaCl2, the contraction induced by caffeine (50 mM) was not significantly altered by LPS treatment . It is concluded that LPS treatment does not reduce the ability of aortic smooth muscle cells to contract . The results suggest that LPS treatment impairs mechanisms involved in calcium handling within smooth muscle cells after stimulation of calcium entry through different pathways and activation of intracellular calcium release by alpha 1-adrenoceptor agonists.

J Mol Biol, 1990 Nov 5, 216(1), 17 - 24
Mutants of pheV in Escherichia coli affecting control by attenuation of the pheS, T and pheA operons . Two distinct mechanisms for de-attenuation; Pages D et al.; Two mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, were previously isolated because they affect attenuator control of the pheS, T operon when the mutant pheV genes are carried by the plasmid pBR322 . We show that the two mutants (A44 and A46) affect attenuator control by different mechanisms . The effect of mutant A44 on pheS, T expression can be progressively decreased by overproduction of Phe-tRNA synthetase, consistent with the mutant tRNA acting as a competitive inhibitor of the enzyme . By contrast, the effect on attenuation of mutant A46 increases with overproduction of Phe-tRNA synthetase, indicating that the mutant must be charged to affect attenuation; we propose that this mutant affects translation directly and causes derepression by competing with wild-type tRNA in translation of the attenuator region leader peptide . Mutant A46 but not mutant A44 leads to further de-attenuation in a miaA background . The presence of two different mechanisms for de-attenuation is further indicated by the finding that a second attenuator controlled by Phe codon translation, from the pheA operon, is affected quite differently by the mutant tRNAs . Finally, experiments involving the introduction of the mutations A44 and A46 into an amber suppressor derived from tRNA(Phe) suggest that both species can function in protein synthesis but with reduced efficiency; mutant A46 is less efficient than mutant A44, consistent with a defect in elongation.

J Biol Chem, 1990 Nov 5, 265(31), 19244 - 8
Aggregated dnaA protein is dissociated and activated for DNA replication by phospholipase or dnaK protein; Hwang DS et al.; dnaA protein isolated from Escherichia coli is equally distributed between a monomeric form, which is active for initiation of DNA replication, and an inactive, aggregated form which contains phospholipids . Replication activity of the aggregated form can be restored by treatments with either dnaK protein or phospholipase A2 . Dissociation of the aggregate by dnaK protein is driven by ATP hydrolysis; action by phospholipase A2 requires a minute concentration of ATP only to stabilize the dissociated protein . Conversion of inactive dnaA-phospholipid complexes to the active form may contribute to the regulation of the initiation of chromosomal replication in E . coli.

J Biol Chem, 1990 Nov 5, 265(31), 18988 - 96
Growth pattern of the murein sacculus of Escherichia coli; Glauner B et al.; The mechanism by which the murein sacculus of Escherichia coli is being enlarged during growth was investigated by pulse and pulse-chase labeling with {3H}diaminopimelic acid . Changes in the composition of the sacculus during aging were analyzed in detail by high performance liquid chromatography separation of the muropeptide subunits released after complete muramidase digestion . After pulses as short as 10 s, a group of novel phosphorylated muropeptides was detected . The kinetics of their appearance is consistent with these structures being derived from the undecaprenylphosphate-linked growing points of murein . A complex maturation process of murein took place including a rapid decay of pentapeptide side chains and a 10-fold increase in tripeptidyl moieties . In addition, the total degree of cross-linkage increased from 16 to 25%, partly due to a 3-fold increase in the formation of LD-A2pm-A2pm cross-links . In pulse-chase experiments the cross-linkage started to decrease after a maximum at about 35 min of chase . The kinetics in the distribution of the radioactivity among acceptor and donor part in the major cross-bridges Tetra-Tetra and Tetra-Tri differed from each other substantially, indicating that the latter structure is completely cleaved within three generations, whereas only 40% of Tetra-Tetra is cleaved during the same time . Furthermore, the attachment of the lipoprotein to murein was delayed by about one generation . It is proposed that these findings reflect an inside-to-outside growth mechanism of the murein sacculus of E . coli.

J Biol Chem, 1990 Nov 5, 265(31), 18968 - 75
Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase . A study using flavin analogs; Prongay AJ et al.; The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138 . Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A . J., engelke, D . R., and Williams, C . H., Jr . (1989) J . Biol . Chem . 264, 2656-2664) . The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD . 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD . The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M . E., and Williams, C . H., Jr . (1984) J . Biol . Chem . 259, 2243-2251) . The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase . The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD . The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3 . The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138 . The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.

J Biol Chem, 1990 Nov 5, 265(31), 18907 - 11
Refolding of an integral membrane protein . OmpA of Escherichia coli; Dornmair K et al.; OmpA is an integral membrane protein from the outer membrane of Escherichia coli . Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS) . Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside . The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy . Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine . Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes . Based on this result, models for sorting of OmpA into the outer membrane of E . coli are discussed.

J Biol Chem, 1990 Nov 5, 265(31), 18860 - 6
Caldesmon-binding sites on tropomyosin; Watson MH et al.; The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin . Binding was examined by fluorescence spectroscopy and affinity chromatography . Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227 . The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200 . This further focused the primary caldesmon-binding site to residues 201-227 . Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon . Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.

J Biol Chem, 1990 Nov 5, 265(31), 18843 - 7
The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli; Shiozuka K et al.; The role of the electrochemical potential difference of proton (delta mu H+) in protein translocation across the membrane of Escherichia coli was examined in detail using an efficient in vitro assay system (Yamada, H., Tokuda, H., and Mizushima, S . (1989) J . Biol . Chem . 264, 1723-1728) . Delta mu H+ reduced the level of ATP necessary for the efficient translocation of OmpF-Lpp, a chimeric model secretory protein . The apparent Km value of the translocation reaction for ATP was lower by 2 orders of magnitude in the presence of delta mu H+ than in its absence . The membrane potential and delta pH, both of which are components of delta mu H+, independently lowered the apparent Km value of the translocation reaction for ATP . An ATP-generating system also lowered the level of ATP required for translocation in the absence of delta mu H+ but not in its presence . It is proposed that ADP formed during protein translocation lowers the affinity of the putative translocation machinery for ATP and that the removal of ADP from the secretory machinery, a possible critical step in the translocation reaction, is stimulated in the presence of either delta mu H+, an ATP-generating system, or a higher concentration of ATP.

J Biol Chem, 1990 Nov 5, 265(31), 18733 - 6
Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli; Melville SB et al.; The fnr gene product, FNR, is a global regulator of anaerobic gene expression in Escherichia coli . When E . coli is switched from aerobic to anaerobic growth conditions, cytochrome o (cyoABCDE) and d oxidase (cydAB) genes are repressed and the anaerobic terminal reductase genes, including nitrate (narGHJI), dimethyl sulfoxide/trimethylamine (dmsABC), and fumarate (frdABCD) reductase, are induced . To determine if certain amino acid residues are essential for FNR to function in this regulatory process, site-directed mutations were introduced into the fnr gene . The resulting mutant proteins were assayed in vivo for their ability to either activate dmsA'-'lacZ and frdA'-'lacZ gene expression, or repress expression of a cyoA'-'lacZ gene fusion . The fnr mutants were grouped into four classes . Class I exhibited a severe decrease in the ability to either activate or repress fnr-dependent gene expression . Mutations in four of the five cysteine residues in the FNR protein were in this class . The sole exception was an FNR Cys16----Ser "mutant" that exhibited normal activity . Class II mutations caused a mild reduction in FNR-dependent activation or repression while Class III mutations conferred a modest increase in the ability of the FNR protein to activate gene expression under aerobic conditions (i.e . FNR*) . Finally, Class IV mutations lowered the modest aerobic FNR transcriptional activation function proportionally more than the anaerobic FNR activity . These findings identify an essential role for the NH2 terminus of the FNR protein in its various activities in anaerobic gene regulation.

J Biol Chem, 1990 Nov 5, 265(31), 18829 - 32
In vitro transposition of transposon Tn3; Ichikawa H et al.; Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract . In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules . Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase . The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3 . The reaction also required DNA synthesis but not RNA synthesis by E . coli RNA polymerase.

J Mol Biol, 1990 Nov 5, 216(1), 85 - 93
Regulation of scallop myosin by mutant regulatory light chains; Goodwin EB et al.; Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC) . Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation . The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains . To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression . Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity . The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity . E . coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus . The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin . A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase . A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues . This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity . Several other point mutations do not alter light chain function . The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site . The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity . The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.

J Mol Biol, 1990 Nov 5, 216(1), 3 - 16
Genomic analysis of sequence variation in tandemly repeated DNA . Evidence for localized homogeneous sequence domains within arrays of alpha-satellite DNA; Warburton PE et al.; As a model to examine the local distribution of sequence variation within large arrays of tandemly repeated DNA in complex genomes, the long-range organization of alpha-satellite DNA from human chromosome 17 was investigated . Three individual chromosomes, representing different alpha-satellite haplotypes, were segregated into mouse and human somatic cell hybrids and their arrays sized by pulse-field gel electrophoresis . An inventory of the higher-order repeat units found in multiple separate regions of these megabase arrays was obtained using cosmid mapping and two-dimensional gel electrophoresis, a technique that combines the large-scale resolution of pulsed-field gel electrophoresis with the small-scale resolution of conventional gel electrophoresis . These analyses show that alpha-satellite arrays are characterized by the presence of localized homogeneous domains containing only one distinct type of repeat unit . These domains, which consist of sequence variants and/or higher-order repeat length variants, can be up to at least several hundred thousands of bases in length . Both abundant and rare variant repeat units can be localized in these distinct domains, which may correspond to transition states in the evolution of tandem multicopy DNA families . This description of the organization of large arrays of tandem repeats provides insight into mechanisms involved in their homogenization.

J Biol Chem, 1990 Nov 5, 265(31), 19249 - 56
Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli . Lysine 893 is critical for activity; Simonin F et al.; Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis . Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA . Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes . In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.

J Biol Chem, 1990 Nov 5, 265(31), 18757 - 61
Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0; Casiano C et al.; Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5 . The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively . The members of the complex were therefore designated Sso L10e and Sso L12e . Sso L12e had other properties in common with E . coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure . The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea . The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e . The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E . coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.

J Biol Chem, 1990 Nov 5, 265(31), 19199 - 207
Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium; Sacchettini JC et al.; Enterocytes located in the pig distal small intestine (ileum) contain a cytosolic protein that is homologous to two proteins that are also synthesized in these cells: intestinal and "liver" fatty acid-binding proteins (I- and L-FABPc, respectively) . To begin to investigate the functional interrelationships of these three proteins, we compared their patterns of tissue-specific expression and developmental regulation in the mouse . Blot hybridization analyses of RNA prepared from 12 adult tissues revealed that this mRNA was confined to the small intestine . Unlike I- and L-FABPc mRNA, which are most abundant in the proximal jejunum, this mRNA is most abundant in the ileum . While I- and L-FABPc gene transcription commence in late fetal life coincident with initial cytodifferentiation of the mouse gut epithelium, the ileal gene is activated later, at the suckling/weaning transition (postnatal day 12) . The ileal location and developmental pattern of expression suggested that this protein may play a role in the intracellular transport of bile salts in the ileal epithelium . To test this hypothesis, we expressed the porcine ileal peptide (PIP) in Escherichia coli, purified it to apparent homogeneity, and analyzed its binding properties for bile acids and fatty acids using 13C NMR spectroscopy . Like I-FABPc, PIP binds palmitate and oleate with a 1:1 molar stoichiometry . However, unlike I-FABPc PIP binds chenodeoxycholate . In addition, the presence of chenodeoxycholate blocks fatty acid binding to PIP, but not to I-FABPc . E . coli-derived PIP was subsequently crystallized with and without chenodeoxycholic acid . All crystals are orthorhombic in the P2(1)2(1)2(1) space group . The unit cell dimensions are a = 36.15 A, b = 50.13 A, and c = 67.18 A.

J Biol Chem, 1990 Nov 5, 265(31), 19185 - 91
A growth factor-inducible nuclear protein with a novel cysteine/histidine repetitive sequence; DuBois RN et al.; Growth factors rapidly induce transcription of a set of genes that encode regulatory proteins, many of which have been identified by cDNA cloning . Here we report the analysis of a cDNA corresponding to a gene induced in mouse 3T3 cells by growth factors and a variety of other extracellular signaling agents . The cDNA encodes a proline-, serine-, and glycine-rich nuclear protein designated Nup475 of 319 amino acids that contains two tandemly repeated cysteine- and histidine-containing sequences (CX8CX5CX3H) suggestive of a novel heavy metal-binding domain . Nup475 produced in Escherichia coli binds zinc . Its mRNA is present in a number of mouse tissues and cell lines, being especially abundant in intestine, thymus, and regenerating liver and in a macrophage cell line stimulated by gamma-interferon . We hypothesize that Nup475 is a regulatory protein with a novel zinc finger structure.

Cell, 1990 Nov 2, 63(3), 619 - 29
Exon mutations uncouple 5' splice site selection from U1 snRNA pairing; Seraphin B et al.; It has previously been shown that a mutation of yeast 5' splice junctions at position 5 (GUAUGU) causes aberrant pre-mRNA cleavages near the correct 5' splice site . We show here that the addition of exon mutations to an aberrant cleavage site region transforms it into a functional 5' splice site both in vivo and in vitro . The aberrant mRNAs are translated in vivo . The results suggest that the highly conserved G at the 5' end of introns is necessary for the second step of splicing . Further analyses indicate that the location of the U1 snRNA-pre-mRNA pairing is not affected by the exon mutations and that the precise 5' splice site is selected independent of this pairing.

Biochim Biophys Acta, 1990 Nov 2, 1029(1), 113 - 6
Lactose transport mutants of Escherichia coli resistant to inhibition by the phosphotransferase system; Wilson TH et al.; The lactose carrier activity of Escherichia coli is inhibited by the binding of dephosphorylated glucose enzyme III . Saier et al . ((1978) J . Bacteriol . 133, 1358-1367) isolated lacY mutants that escaped this inhibition . This communication reports the cloning and sequencing of one of the Saier mutants and the isolation, cloning and sequencing of another similar mutant . Both mutations resulted in amino acid substitutions on the middle cytoplasmic loop of the carrier (alanine-198 to valine and serine-209 to isoleucine) . It is concluded that this cytoplasmic loop may be one of the sites of binding of glucose enzyme III.

Anal Biochem, 1990 Nov 1, 190(2), 331 - 9
Detection of protein-DNA complex formation by time-resolved fluorescence depolarization of bound ethidium bromide; Cook J et al.; We introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and DNA . Changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to DNA segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and DNA . We have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific DNA-binding proteins involved in transcriptional regulation (the cro repressor of coliphage lambda, the lac repressor of Escherichia coli, and the RNA polymerase of coliphage T7) and their target DNA fragments ranging in length from 17 to 36 base pairs . The technique allows one to follow complex formation while varying solution conditions such as temperature, pH, ionic strength, and presence of effector molecules . Macromolecular concentrations ranging from 10(-7) to 10(-4) M can be used, allowing estimates of relative binding affinities . The magnitude of the observed rotational correlation times (phi obs) can be used to infer information about the size and shape of the complexes.

Photochem Photobiol, 1990 Nov, 52(5), 1017 - 23
Photosensitized damage to supercoiled plasmid DNA induced by 334-nm radiation in the presence of 2-thiouracil consists of alkali- and piperidine-labile sites as well as frank strand breaks; Churchill ME et al.; A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura) . After irradiation, some DNA samples were treated to reveal labile sites . Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning . Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS) . The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation . Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types . The relative proportions of the three lesion types produced by several DNA damaging treatments were measured . Hydrogen peroxide, gamma-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively . Treatment with singlet oxygen {data from Blazek et al . (1989) Photochem . Photobiol . 48, 607-613} produced much different proportions, with a PLS:FSB ratio of 4.1:1 . These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.

Kansenshogaku Zasshi, 1990 Nov, 64(11), 1454 - 61
{Enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila using 60-kDa protein antigen}; Morimoto T; The enzyme-linked immunosorbent assay (ELISA) has been evaluated for the detection of antibodies against Legionella pneumophila . Three-grade antigens were prepared from Legionella pneumophila serogroup I . Crude antigen was made by enzyme digestion, sonication and centrifugation and then became half pure by ammonium sulfate precipitation . It was purified to form 60-kDa protein antigen by size-exclusion chromatography on a Sephacryl S400 column and ion-exchanged chromatography on a DEAE-5PW column . 60-kDa protein antigen was the most sensitive of the three antigens, but more cross reactive to K . pneumoniae type II than the other two antigens . It is suggested that crossreaction occurs on the grounds whether 60-kDa protein is antigen common to L . pneumophila serogroup I and K . pneumoniae type II or antigens of such two bacteria co-exist on 60-kDa protein.

J Burn Care Rehabil, 1990 Nov-Dec, 11(6), 531 - 7
The effect of thromboxane synthetase inhibition on cardiopulmonary function during endotoxemia in sheep; Fujioka K et al.; The early pulmonary hypertension seen with endotoxin (lipopolysaccharide) has been reported as resulting from the release of thromboxane A2 . We studied the cardiopulmonary response to endotoxin in sheep with and without treatment with a thromboxane synthetase inhibitor, OKY-046 . The animals were implanted with instruments for crystallographic dimension analysis of the left ventricle and measurement of left ventricular, aortic, left atrial, and pulmonary arterial pressures and cardiac index . Thirteen sheep received 1.0 micrograms/kg of Escherichia coli endotoxin with (n = 6) and without (n = 7) OKY-046 (10 mg/kg bolus, then 10 micrograms/kg/min) . OKY-046 prevented the increase in pulmonary arterial pressure and decrease in cardiac index usually seen during the early phase of endotoxemia . Between 8 and 12 hours after the administration of endotoxin, cardiac index increased from 6.4 +/- 0.8 to 8.4 +/- 0.8 L/min/m2 . Concomitantly, the end-systolic pressure/diameter relationship (a sensitive myocardial contractility index) significantly decreased from 14.7 +/- 0.6 to 7.7 +/- 0.7 mm Hg/mm . Another index of the left ventricular contractility, the maximum rate of pressure rise was also reduced . OKY-046 prevented decreases in end-systolic pressure/diameter relationship and maximum rate of pressure rise.

Br J Clin Pract, 1990 Nov, 44(11), 514 - 5
Endocarditis, pyrexia of unknown origin and occult abdominal malignancy; Kounis NG et al.; Two patients who presented with pyrexia of unknown origin were found to have carcinoma of the caecum without gastrointestinal symptoms . Blood cultures were positive for Escherichia coli, and in one patient the diagnosis of endocarditis was confirmed by echocardiography . This rare association may be fortuitous, but a common pathogenetic basis cannot be excluded.

Pathol Biol (Paris), 1990 Nov, 38(9), 928 - 34
{Age-related changes in plasma androstanediol glucuronide in men}; Nahoul K et al.; Plasma 5 alpha-androstane-3 alpha, 17 beta diol (Adiol) glucuronide levels were determined using a reliable radioimmunoassay involving hydrolysis with E . coli beta-glucuronidase, diethylether extraction, and Celite column chromatography . Conditions ensuring optimal hydrolysis were determined as well as quality control criteria . Sixty-eight male out-patients wre studied . These subjects were divided into three age groups: I (n = 21; age 20-35 years), II (n = 21; age: 36-50 years), and III (n = 26; age: 51-70 years) . Patients under 50 years of age had infertility with no abnormalities upon sperm analysis . Older patients had erectile dysfunction . All subjects had serum gonadotropin (FSH and LH) and prolactin levels within the normal range for age . Adiol glucuronide levels (mean +/- SD) were as follows: 5.0 +/- 2.2 ng/ml (range: 1.6-9.5), 5.4 +/- 3.8 ng/ml (range 1.4-16.0) and 4.5 +/- 2.5 ng/ml (range 1.2-9.3) in groups I, II and III respectively . A trend towards lower Adiol glucuronide levels with advancing age was found but there was no significant difference between group III and the other two groups . Similarly, no significant correlation was found between Adiol glucuronide levels and age (r = -0.12) . Conversely, a significant correlation (r = 0.37; p less than 0.01) was observed between Adiol glucuronide levels and bioavailable testosterone (T) levels . This finding might be the consequence of a certain enhancement of 5 alpha-reductase activity with age and/or the non significant decrease with age of another precursor.

Mol Gen Genet, 1990 Nov, 224(2), 232 - 40
Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina; Sellem CH et al.; In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains . In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron . This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli . We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures . A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

Mol Gen Genet, 1990 Nov, 224(2), 209 - 21
The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns; Labouesse M; The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS) . Herbert et al . (1988, EMBO J 7:473-483) proposed that this protein is involved in mitochondrial RNA splicing . Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene . Three of these prevent respiration while maintaining the mitochondrial genome . These three mutants: (1) display in vitro a mLRS activity ranging from 0%-50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3 . Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2- mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase . We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.

Mol Gen Genet, 1990 Nov, 224(2), 201 - 8
Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins; Hess J et al.; We studied the efficiency of the pHly152-derived haemolysin transport system using PhoA-HlyA fusion proteins and different constructs which provide HlyB/HlyD in trans . The optimal C-terminal HlyA signal consists of the last 60 amino acids . Longer stretches of HlyA do not improve the transport efficiency of PhoA-HlyA fusion proteins . The introduction of deletions and/or replacements in the 60 amino acid HlyA signal domain revealed at least three functional regions with different degrees of specificity . Amino acids 1-21 (numbered from the N-terminal part of the 60 amino acid HlyA signal), termed region I, could be replaced by a Pro-containing peptide . The other two regions II and III (amino acids 22-40 and 41-60, respectively) seem to interact directly with the HlyB/HlyD translocator since a PhoA fusion protein which contains either of the two regions was still secreted in a HlyB/HlyD-dependent mode, albeit at low efficiency . An efficient trans-complementing HlyB/HlyD system was only obtained from the pHLy152-encoded hly determinant when the regulatory hlyR element was provided in cis . Secretion of the PhoA-HlyA fusion protein did not interfere with the secretion of HlyA even when the fusion protein was induced to a high level . This suggests that the capacity of the HlyB/HlyD translocation system is high and not normally saturated by its natural HlyA substrate.

Mol Gen Genet, 1990 Nov, 224(2), 169 - 76
Proteolytic processing of MucA protein in SOS mutagenesis: both processed and unprocessed MucA may be active in the mutagenesis; Shiba T et al.; The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals . Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis . By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB . Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ'-'MucA fusion protein . Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion . In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis . However, MucA was not processed in the recA430 cells treated with mitomycin C . We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26) . MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA . The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA+ in UV mutagenesis . These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.

Int J Parasitol, 1990 Nov, 20(7), 943 - 50
A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs; Gasser RB et al.; Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens . These antibodies and serum pools derived from dogs with E . granulosus infections were used to screen a lambda gt11 cDNA library constructed using E . granulosus protoscolex mRNA . Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E . granulosus infected dogs . These clones were all subcloned into the plasmid vector pGEX-1 . Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot . Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E . granulosus, Taenia spp . or nematodes, and helminth-free dog sera . The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E . granulosus infection in dogs despite its relatively low sensitivity . Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.

Genes Dev, 1990 Nov, 4(11), 1899 - 909
A plant DNA-binding protein increases the number of active preinitiation complexes in a human in vitro transcription system; Katagiri F et al.; TGA1a is a tobacco DNA-binding protein that binds to the activation sequence-1 (as-1) element of the cauliflower mosaic virus 35S promoter . We have produced TGA1a in Escherichia coli, purified it from bacterial extracts, and examined its effect on transcription in a human in vitro system . Addition of TGA1a stimulates transcription by up to 20 times, and the stimulation is dependent on the presence of the as-1 element in the promoter . When transcription reinitiation is inhibited by 0.3 M KCl, activation is similar . Therefore, TGA1a activates transcription by increasing the number of active preinitiation complexes . After formation of the preinitiation complexes in the presence of TGA1a, oligonucleotides containing TGA1a-binding sites do not significantly affect the stimulated level of transcription . This result indicates that a complex remains committed to the promoter site after initiation and that this complex is used repeatedly during several initiation events . Our study demonstrates for the first time that a plant factor can activate transcription in a human in vitro system and that the activation mechanism of the plant factor is similar to that of mammalian factors.

J Dairy Sci, 1990 Nov, 73(11), 3126 - 31
Hemagglutination and hemolysis by Escherichia coli isolated from bovine intramammary infections; Hogan JS et al.; Seventy-six Escherichia coli isolated from bovine intramammary infections were tested for hemagglutination and hemolysis of erythrocytes . Fifty-seven percent of isolates were hemagglutination-positive for bovine erythrocytes compared with 46% that agglutinated guinea pig erythrocytes . Twenty-eight percent of isolates were hemagglutination-positive for erythrocytes from both species . Only 14.5 and 2.6% of isolates were mannose-resistant, hemagglutination-positive for bovine and guinea pig erythrocytes, respectively . Neither duration nor severity of infection from which isolates were obtained differed between isolates that were hemagglutination-positive and hemagglutination-negative . Percentage distribution of hemagglutination-positive isolates did not differ among isolates from infections that originated at calving, during lactation, or the first half of the dry period . Hemagglutination reactions were also not related to in vitro growth in cell-free dry cow secretion . Percent of isolates that caused hemolysis of washed bovine erythrocytes was 2.6% compared to 3.9% for sheep erythrocytes . Hemolysis was not related to hemagglutination . Hemagglutination and hemolysis of erythrocytes did not appear to be virulence factors for E . coli isolated from bovine intramammary infections.

Biotechniques, 1990 Nov, 9(5), 598 - 606
Sub-femtomole quantitation of proteins with Threshold, for the biopharmaceutical industry; Briggs J et al.; The Threshold system provides for rapid quantitation of a variety of analytes at sub-femtomole levels, which is fewer than 6 x 10(8) molecules . The operating principles of the measurement system will be described, including the means for chemical modulation of the signal and the subsequent signal detection utilizing a proprietary silicon sensor . The Immuno-Ligand Assay is a universal ligand-binding assay system which provides quantitative measurement of proteins (including antibodies) in a variety of biological media . Assay performance will be described, along with data demonstrating sensitivity, precision and accuracy, for a variety of analytes of interest to the biopharmaceutical industry.

Biotechniques, 1990 Nov, 9(5), 538 - 40
Rapid isolation of substrate-quality plasmid DNA without CsCl-dye density gradients; Aguilar-Cordova E et al.; The isolation of plasmid DNA produced in transformed bacterial cells is essential for many molecular biology techniques . Two drawbacks to the widely used CsCl-ethidium bromide method of preparation are the need for ultracentrifuge time and the generation of ethidium bromide waste . In this article we describe a method for the quick isolation of plasmid DNA without the use of an ultracentrifuge or ethidium bromide.

Res Vet Sci, 1990 Nov, 49(3), 349 - 54
Toxic effects for lambs of cytotoxic necrotising factor from Escherichia coli; De Rycke J et al.; The lethal effect, clinical signs and lesions caused by the intravenous inoculation into six lambs (seven to 10 days old) of a partly purified preparation of cytotoxic necrotising factor (CNF) from Escherichia coli, strain BM2-1, were investigated . Two control preparations were also tested in two lambs each: (i) the same as above but heated for one hour at 60 degrees C, a treatment which inactivates CNF and preserves residual endotoxic activity; and (ii) purified material from a CNF-defective mutant of BM2-1 . Whereas none of the lambs in either of the control groups died or showed significant clinical signs or lesions, all the lambs inoculated with partly purified CNF developed severe clinical signs starting six hours after inoculation which consisted mainly of neurological signs and mucoid diarrhoea . The most striking lesions were oedema and haemorrhages in the central nervous system, and foci of coagulation necrosis in the myocardium . Mucus hypersecretion in the gastrointestinal tract was not associated with cellular inflammation.

Res Vet Sci, 1990 Nov, 49(3), 292 - 7
Intravenous solutions for fluid therapy in calf diarrhoea; Groutides CP et al.; Five commercially available parenteral solutions were compared for their effectiveness in correcting the disturbances associated with diarrhoea induced by Escherichia coli . Each solution (saline, Hartmann's, Darrow's, Plasmalyte +/- glucose) was tested on eight Jersey calves less than a week old and weighing approximately 25 kg . Each calf received 8.5 litres over three days, at about 20 ml kg-1 h-1 . Solutions such as saline or Plasmalyte which had higher concentrations of sodium were more effective at correcting dehydration and electrolyte disturbances than those with less sodium (Darrow's, Hartmann's) but only those with bicarbonate precursors (lactate, acetate, gluconate) were effective in correcting metabolic acidosis . The additional potassium in Darrow's was predictably unhelpful in correcting hyperkalaemia and the additional glucose in Plasmalyte-glucose, despite some beneficial effects, undermined its effectiveness in correcting acidosis . These results suggest that solutions for intravenous treatment should probably contain about 150 mmol litre-1 Na+, 5 mmol litre-1 K+ and about 50 mmol litre-1 of a mixture of bicarbonate and precursors . Neither of the commonly used solutions (saline or Hartmann's) is thus ideal.

DNA Cell Biol, 1990 Nov, 9(9), 631 - 5
Nucleotide sequence and deletion analysis of the polB gene of Escherichia coli; Chen H et al.; The polB gene encodes DNA polymerase II in Escherichia coli . The nucleotide sequence shows an open reading frame of 2,304 nucleotides coding for a protein of 88 kD . The protein initiation signal is preceded by a lexA box lying 2 nucleotides from the termination signal of araD, and begins with GUG 75 nucleotides after the termination of araD . The polB gene and the araD gene are transcribed in the same direction . Initiation of protein synthesis was confirmed by peptide sequence . We have also demonstrated that the polB sequence is lacking in some strains . We conclude that DNA polymerase II is not a required protein in the cell . Sequence comparisons show that DNA polymerase II is an alpha-like DNA polymerase.

Eur J Immunol, 1990 Nov, 20(11), 2479 - 84
Autoimmune reactions to heat-shock proteins in pristane-induced arthritis; Thompson SJ et al.; The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil, 2,6,10,14-tetramethylpentadecane (pristane), was shown to depend on an intact immune response possibly to a heat-shock protein (hsp) in the synovium . Initial experiments suggested that some crucial event in the development of arthritis takes place early after pristane injection . First, irradiated pristane-treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors within 25 days of irradiation . Second, mice irradiated up to 50 days after pristane injection, but not later, did not develop arthritis . Evidence for the involvement of an immune response to heat-shock protein (hsp) comes from the finding that mice injected with mycobacterial 65-kDa hsp prior to pristane challenge had a reduced incidence of arthritis in contrast to animals pre-immunized with the E . coli hsp equivalent GroEL or with bovine serum albumin . Other experiments revealed that T cells from mice with gross morphologically defined arthritis proliferated strongly to hsp65 and to normal joint antigens, whereas T cells from animals treated with pristane which did not develop arthritis gave much smaller responses . Mice which developed arthritis also had elevated levels of anti-hsp65 IgG in comparison with non-arthritic animals . These findings strongly suggest that autoimmune reactions to an antigen which cross-reacts with hsp65 are generated in pristane-induced arthritis . It is considered that the autoimmune response is directed to a synovial antigen and that pre-immunization with hsp65 protects the animals from the development of pristane-induced arthritis by altering the specificity or quality of the immune response to this antigen.

Genetics, 1990 Nov, 126(3), 519 - 33
Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli; Stahl FW et al.; When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture . Otherwise, the chi 0 recombinant is recovered in excess . This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant . The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid . When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated . These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination . The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences . (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8955 - 9
Expression of the archaebacterial bacterio-opsin gene with and without signal sequences in Escherichia coli: the expressed proteins are located in the membrane but bind retinal poorly; Karnik S et al.; In a further effort to obtain functional expression of the bacterio-opsin gene (bop) in Escherichia coli, the bop gene with E . coli signal sequences as well as the bop gene with the native presequence were expressed in E . coli . The location of the expressed products in the E . coli cell and their processing and folding to a structure that binds retinal as in Halobacterium halobium were investigated . All the expressed proteins were in the membrane . The proteins were largely unprocessed, and they were distributed between the outer and the inner membrane . The processed fractions, which were minor, were exclusively in the inner membrane . The processed proteins bound exogenously added all-trans-retinal but only partially, indicating that these proteins were present in at least two folded states.

Mol Immunol, 1990 Nov, 27(11), 1113 - 8
Inhibition of in vitro transcription by adenosine antibodies; Vaishnav YN et al.; Antibodies against adenosine markedly inhibited in vitro transcription in isolated BHK 21 nuclei in a dose-dependent manner . The inhibition was specific as it could be completely reversed by the addition of homologous hapten . Addition of RNA at low concentration reversed the inhibition, whereas excess DNA did not have any effect . Adenosine antibodies also inhibited in vitro transcription with calf thymus DNA and E . coli RNA polymerase . Antibodies that react with DNA but not with RNA such as anti-dpA, anti-dpC and anti-DNA failed to inhibit in vitro transcription in isolated nuclei as well as with calf thymus DNA and E . coli RNA polymerase . The results strongly indicate that the binding of adenosine antibodies to RNA is responsible for the inhibition of transcription.

J Surg Res, 1990 Nov, 49(5), 413 - 8
Immunorestorative effects of reimplanted splenic tissue and splenosis; Ludtke FE et al.; Different immune functions were analysed in detail in 41 patients who had been splenectomized after a traumatic rupture of the spleen within four years after surgical intervention . Patients were assigned to one of the following groups as judged by liver/spleen scintigraphy: (1) patients with reimplanted splenic tissue, (2) patients with splenosis, and (3) patients without splenic tissue . Leukocytosis and an increased number of total lymphocytes as well as B-cells were observed in patients of all groups . In addition, the number of circulating T-suppressor cells was significantly increased in patients with no detectable splenic tissue . In contrast, serum concentrations of immunoglobulins and complement components were in the normal range; similarly, phagocytosis-associated functions of the patients' neutrophils and monocytes were found to be unimpaired (chemiluminescence and particle uptake) . However, in all groups of splenectomized patients a deficiency in specific serum opsonic activity against a strain of Escherichia coli (O:102, H:6) could be detected . We conclude that neither splenosis nor autologous reimplantation of splenic tissue restores opsonic deficiency caused by splenectomy.

J Clin Invest, 1990 Nov, 86(5), 1511 - 6
Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria; Delfau MH et al.; Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector . Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein . Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme . One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

Arch Biochem Biophys, 1990 Nov 1, 282(2), 233 - 8
Variations in the activity of glutathione reductase and the cellular glutathione content in relation to sensitivity to methylviologen in Escherichia coli; Kunert KJ et al.; To study the function of glutathione reductase and glutathione in Escherichia coli the coding sequence of the bacterial glutathione reductase gene (gor gene) was cloned into the vector pBR322, and the gor gene was expressed under the control of the promoter of the tetracycline-resistance gene (tet gene) in different Escherichia coli strains . Cells of the gor-mutant strain SG5 containing the vector pBR322 (SG5:pBR322) had no detectable glutathione reductase activity and a significantly lower total glutathione (GSH + GSSG) content relative to control cells of the strain JM101 (JM101: pBR322) . The gor mutant cells were less sensitive to inhibition by methylviologen (as defined by changes in growth) than cells of the strain JM101 . Elevated levels of both glutathione reductase activity and the total glutathione content (GSH + GSSG) were found when the gor gene was expressed in cells of the gor-mutant strain SG5 (SG5:pJIK1) . Thus the activity of glutathione reductase is essential in order to maintain a high glutathione content . Furthermore, cells of the strain SG5: pJIK1 showed an increased sensitivity to methylviologen compared to cells of the gor mutant containing the vector pBR322 alone without the cloned gor gene insert (SG5:pBR322) . In all experiments, the glutathione pool (GSH + GSSG) of bacterial cells was 90% reduced . In methylviologen-sensitive sodB mutant cells lacking iron superoxide dismutase activity (QC773:pBR322) overexpression of the cloned gor gene resulted in an elevated level of glutathione reductase activity which partially protected sodB mutant cells (QC773:pJIK1) against methylviologen toxicity . In sodB mutant cells expressing the gor gene (QC773:pJIK1) protection by glutathione reductase was, however, less effective than protection provided by expression of the iron superoxide dismutase gene (sodB gene) in these mutant cells (QC773:pJIK2) . In sodA mutant cells lacking manganese superoxide dismutase activity but expressing the cloned gor gene (QC772:pJIK1) increased cellular glutathione reductase activity did not provide protection against methylviologen.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8521 - 5
Expression of fully functional tetrameric human hemoglobin in Escherichia coli; Hoffman SJ et al.; Synthetic genes encoding the human alpha- and beta-globin polypeptides have been expressed from a single operon in Escherichia coli . The alpha- and beta-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to greater than 5% of the total cellular protein . Purified recombinant hemoglobin has the correct stoichiometry of alpha- and beta-globin chains and contains a full complement of heme . Each globin chain also contains an additional methionine as an extension to the amino terminus . The recombinant hemoglobin has a C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE . The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0 . The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains . We have also expressed the alpha- and beta-globin genes separately and found that the expression of the alpha-globin gene alone results in a marked decrease in the accumulation of alpha-globin in the cell . Separate expression of the beta-globin gene results in high levels of insoluble beta-globin . These observations suggest that the presence of alpha- and beta-globin in the same cell stabilizes alpha-globin and aids the correct folding of beta-globin . This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8467 - 71
Architecture of ribosomal RNA: constraints on the sequence of "tetra-loops"; Woese CR et al.; The four-base loops that cap many double-helical structures in rRNA (the so-called "tetra-loops") exhibit highly invariant to highly variable sequences depending upon their location in the molecule . However, in the vast majority of these cases the sequence of a tetra-loop is independent of its location and conforms to one of three general motifs, GNRA, UNCG, and (more rarely) CUUG . For the most frequently varying of the 16S rRNA tetra-loops, that at position 83 (Escherichia coli numbering), the three sequences CUUG, UUCG, and GCAA account for almost all examples encountered, and each of them has independently arisen at least a dozen times . The closing base pair of tetra-loop hairpins reflects the loop sequence, tending to be C.G for UUCG loops and G.C for CUUG loops.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8242 - 6
Amplification of large artificial chromosomes; Smith DR et al.; Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated . However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome . We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes . The vector includes a conditional centromere that can be turned on or off by changing the carbon source . Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene . When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold . The selective conditions did not induce obvious rearrangements in any of the clones tested . Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations . Applications of copy number control to various aspects of artificial chromosome analysis are addressed.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8222 - 6
Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C base pairs; Petracek ME et al.; We have isolated telomeric DNA and telomere-associated sequences from Chlamydomonas reinhardtii . The terminal telomere sequences of the green alga Chlamydomonas are composed of (TTTTAGGG)n repeats that are similar, but not identical, to those of the higher plant Arabidopsis thaliana . We demonstrate that these repeats are telomeric by their preferential sensitivity to nuclease Bal-31 digestion, their similarity to A . thaliana telomeres, their orientation relative to the end of the chromosome, and the methods used for their isolation . Five independent telomere clones were isolated, and three of these clones include closely related telomere-associated sequences . One of these telomere-associated sequences hybridizes to a number of genomic fragments sensitive to digestion with the exonuclease Bal-31 . Like telomere sequences from other organisms, the C . reinhardtii telomeres display a bias for guanine and thymine nucleotides on the 3'-end strand . However, the sequence of Chlamydomonas telomeres is more A + T-rich than any other known telomere sequence . We propose that the common feature of all known telomere is the frequent occurrence of tracts of three or more adjacent guanine residues.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8202 - 6
Interchangeable RNA polymerase I and II enhancers; Lorch Y et al.; The RNA polymerase I (pol I) enhancer of Saccharomyces cerevisiae contains at least three elements commonly associated with RNA polymerase II (pol II) enhancers, binding sites for the transcriptional activators general regulatory factor 2 and autonomously replicating sequence-binding factor I, and a thymidine-rich element . When the particular form of the thymidine-rich element found in the pol I enhancer was placed in front of a pol II promoter, transcription was stimulated 43-fold, comparable to the effect of a powerful pol II activator such as Gal4 . Conversely, when two copies of a thymidine-rich element from a pol II enhancer were placed upstream of a pol I promoter, transcription was stimulated 38-fold . This functional reciprocity of pol I and II enhancers may reflect similarities in the mechanisms of transcriptional activation . The pol I enhancer also contains an element that appears to be pol I-specific and prevent the activation of pol II.

Nature, 1990 Nov 1, 348(6296), 86 - 8
A downstream initiation element required for efficient TATA box binding and in vitro function of TFIID; Nakatani Y et al.; The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells . In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint . One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site . We have now shown by transfection that both elements are required for efficient transcription in cultured cells . A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator . Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro . In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region . The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription . These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

Mutat Res, 1990 Nov-Dec, 233(1-2), 95 - 103
N-methyl-N'-nitro-N-nitrosoguanidine induced DNA sequence alteration; non-random components in alkylation mutagenesis; Gordon AJ et al.; Our approach to the study of how the molecular nature of DNA modulates the behavior of mutational sites involves the characterisation of distributions of mutations . The Escherichia coli lacI genetic/M13 cloning system allows the comparison of base substitution frequencies at a large number of sites . The observed distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced G:C----A:T transition (the predominant event), and A:T----G:C transition (a relatively rare event), is strikingly non-random . Some sites of G:C----A:T mutation are almost 100 times more often mutated by MNNG than the least susceptible sites . Sites of mutation, however, do not display a continuum of mutability, but rather can be strictly demarcated by their 5' flanking base . Sites with a high frequency of occurrence share a common sequence motif, namely 5'-R-G-N-3', which is the sole apparent feature that distinguishes them from sites less commonly mutated (i.e . 5'-Y-G-N-3') . A corollary of this defined site specificity is the absence of a strand bias in MNNG-induced lacI-d mutation . The availability of specific or non-specific alkylation-repair systems does not appear to alter the distribution of mutation, which suggests that the observed mutational distribution is a direct reflection of the initial damage distribution . MNNG does not belong to that class of compounds typified by ultraviolet light or 4-nitroquinoline-N-oxide which exhibit both random and non-random components of mutagenesis.

Mol Cell Biol, 1990 Nov, 10(11), 5707 - 20
The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication; Gibson SI et al.; MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells . It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein . We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1 . The mcm3-1 mutant was found to be thermosensitive for growth . Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination . Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n . These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis . The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

J Infect Dis, 1990 Nov, 162(5), 1075 - 80
Fucosylated oligosaccharides of human milk protect suckling mice from heat-stabile enterotoxin of Escherichia coli; Newburg DS et al.; Human milk protects suckling mice from the diarrheagenic effects of heat-stabile enterotoxin of Escherichia coli (ST) . To identify the human milk fraction responsible for this protection, pooled skimmed, deproteinated milk was passed through charcoal, whereupon lactose was separated from the oligosaccharides . The oligosaccharides contained ST-protective activity; the lactose did not . The neutral, but not the acidic, fraction exhibited protective activity against ST (22% vs . 57% mortality, respectively; P less than .001) . The fucosylated, but not the nonfucosylated, subfractions of the neutral fraction contained the factor protective against ST (35% vs . 50% mortality, respectively; P less than .05) . An oligosaccharide isolation scheme based on different principles produced confirmatory results . The commercially available neutral fucosylated oligosaccharides of human milk did not significantly protect the mice from the effects of ST . Thus, the protective factor against ST seems to be a minor neutral fucosyloligosaccharide of human milk.

J Infect Dis, 1990 Nov, 162(5), 1069 - 74
Properties of strains of Escherichia coli O26:H11 in relation to their enteropathogenic or enterohemorrhagic classification; Scotland SM et al.; Thirty-seven strains of Escherichia coli O26:H11 from infants and calves with diarrhea were examined for properties associated with enteropathogenic (EPEC) or enterohemorrhagic E . coli (EHEC) . Strains were heterogeneous with respect to Vero cytotoxin (VT) production and hybridization with the EHEC plasmid-specific (CVD419) probe; 26 strains produced VT1; 1 produced VT2 . Twenty-four of 27 VT+ strains and 5 of 10 VT- strains hybridized with the CVD419 probe and produced enterohemolysin; these properties are characteristic of EHEC . The strains did not hybridize with the EPEC adherence factor probe, a property characteristic of some EPEC . Nevertheless, 36 strains adhered to HEp-2 cells in a localized manner and were positive by the fluorescence actin staining (FAS) test that is considered to correlate with the ability to cause attaching and effacing lesions in vivo . EPEC and EHEC cause these lesions . Although the FAS test appeared to be the most general pathogenicity test for the O26:H11 strains, it could not be used to assign strains specifically to EPEC or EHEC groups.

J Bacteriol, 1990 Nov, 172(11), 6611 - 4
Regulation of expression of the ftsA cell division gene by sequences in upstream genes; Dewar SJ et al.; The essential cell division genes ftsQ and ftsA overlap by 1 bp (A . C . Robinson, D . J . Kenan, G . F . Hatfull, N . F . Sullivan, R . Spiegelberg, and W . D . Donachie . J . Bacteriol . 160:546-555, 1984; Q.-M . Yi, S . Rockenbach, J . E . Ward, and J . F . Lutkenhaus . J . Mol . Biol . 184:399-412, 1985) . We have previously shown that ftsA can be expressed from a weak promoter located within the ftsQ gene (Robinson et al., J . Bacteriol . 160:546-555, 1984) . We report here the effects on ftsA expression of a series of deletions within ftsQ . We find that two regions upstream of the promoter are important in its expression . When both are present, ftsA is expressed, as is also the case when both are absent . The two regulatory elements (O1 and O2) have 9-bp sequences, of which 8 bp are identical.

J Bacteriol, 1990 Nov, 172(11), 6573 - 5
Biosynthesis of a membrane adhesion zone fraction throughout the cell cycle of Escherichia coli; Joseleau-Petit D et al.; Synchronized cells of Escherichia coli were pulse-labeled with {3H}leucine and subjected to membrane fractionation to determine whether a fraction that is enriched for membrane-murein adhesion zones (fraction OML) was preferentially generated at specific times during the cell cycle, as previously suggested from studies of lkyD and cha mutants . Contrary to this prediction, the experiments showed that OML was formed continuously during the division cycle.

J Bacteriol, 1990 Nov, 172(11), 6452 - 8
Interaction of RecA protein with acidic phospholipids inhibits DNA-binding activity of RecA; Krishna P et al.; The RecA protein of Escherichia coli binds specifically to acidic phospholipids such as cardiolipin and phosphatidylglycerol . This binding appears to be affected by the presence of divalent cations such as Ca2+ and Mg2+ . The interaction leads to the inhibition of RecA binding to at least two different conformations of DNA, single-stranded DNA and left-handed Z-DNA, thus suggesting that the phospholipids interact at the DNA-binding site of the RecA protein . Inclusion of a nucleotide cofactor {adenosine 5'-O-(gamma-thiotriphosphate)} in the reactions did not prevent the inhibition of DNA-binding activities of RecA protein by the phospholipids . The interaction of RecA protein with cardiolipin and phosphatidylglycerol, which represent two of the three major phospholipids of the E . coli membrane, may be physiologically important, as it provides a possible mechanism for the RecA-membrane association during the SOS response . These observations raise the possibility that the Z-DNA-binding activity of RecA protein is merely a manifestation of its phospholipid-binding property.

J Bacteriol, 1990 Nov, 172(11), 6386 - 95
Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli; Sung LM et al.; Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli . Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon . Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene . The slt-IIv promoter was demonstrated to be identical to the slt-II promoter . The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters . The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter . In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E . coli strains with wild-type or mutant ferric uptake regulation (fur) alleles . Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene . A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB . These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron . Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.

J Bacteriol, 1990 Nov, 172(11), 6300 - 7
Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2; Amemura M et al.; Transcription of the genes in the phosphate regulon in Escherichia coli is activated by PhoB protein, which is phosphorylated by PhoR protein under phosphate-limiting conditions . In the absence of the phoR function, the genes in the phosphate regulon are expressed constitutively and the expression is dependent on the function of phoM and phoB . We constructed a plasmid with a lacZ'-'phoM fusion gene, which encoded a hybrid protein (PhoM1206) in which the hydrophobic amino-terminal half of the native PhoM was replaced by beta-galactosidase . The phoM1206 gene could complement the phoM mutation in vivo . We purified PhoM1206 from the overproducing strain carrying the plasmid; it was autophosphorylated at a histidine residue in the presence of ATP, and the phospho-PhoM1206 phosphorylated PhoB . PhoM1206 could also transphosphorylate the product of phoM-orf2, which is structurally homologous to phoB and located immediately upstream of phoM . Although PhoR1084 that lacked the hydrophobic amino-terminal region of the native PhoR protein transphosphorylated PhoB, it could not phosphorylate PhoM-open reading frame 2 . Therefore, cross talk by protein phosphorylation appears to occur from PhoM to PhoB but not from PhoR to PhoM-open reading frame 2.

J Bacteriol, 1990 Nov, 172(11), 6291 - 9
Purification and preliminary characterization of the Escherichia coli K-12 recF protein; Griffin TJ 4th et al.; The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair . To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product . The RecF protein was overproduced and purified to apparent homogeneity . N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present . The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays . Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM . The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity . Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.

J Bacteriol, 1990 Nov, 172(11), 6268 - 73
The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system; Iwasaki H et al.; The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the polB gene encoding DNA polymerase II was also mapped . We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome . The cells that harbored multicopy plasmids with the dinA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II . Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase . From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system . The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method.

J Bacteriol, 1990 Nov, 172(11), 6223 - 31
LexA-independent expression of a mutant mucAB operon; McNally KP et al.; pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis . Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor . pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate . In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host . The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region . We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites . DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA . This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence . The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.

Infect Immun, 1990 Nov, 58(11), 3717 - 23
Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli; Fasano A et al.; It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin . We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins . All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E . coli . Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance . Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production . Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.

Infect Immun, 1990 Nov, 58(11), 3645 - 52
Construction of a bifunctional Escherichia coli heat-stable enterotoxin (STb)-alkaline phosphatase fusion protein; Urban RG et al.; A fusion between the genes encoding the Escherichia coli STb heat-stable enterotoxin (estB) and alkaline phosphatase (phoA) was constructed, and the expressed protein product was characterized . The STb-alkaline phosphatase protein (STb-PhoA) had an apparent molecular mass of 50,000 daltons and was detected with both monoclonal anti-alkaline phosphatase and polyclonal anti-STb antibodies . Expression of the gene fusion resulted in high-level production of alkaline phosphatase activity, indicating that STb-PhoA was processed and exported into the periplasm of the E . coli host strain . Amino acid sequence analysis of the hybrid protein yielded the sequence Ser-Thr-Gln-Ser-Asn-Lys-Lys, indicating that STb-PhoA was processed during export in a fashion identical to that of native STb (Y . M . Kupersztoch, K . Tachias, C . R . Moomaw, L . A . Dreyfus, R . G . Urban, C . Slaughter, and S . Whipp, J . Bacteriol . 172: 2427-2432, 1990) . STb-PhoA was purified from an expressed bacterial lysate by preparative isoelectric focusing . In a rat ligated intestinal loop model, purified STb-PhoA induced highly significant (P less than 0.002) fluid secretion . In addition, the specific activity of STb-PhoA was nearly identical to that of purified STb . Thus, the STb-PhoA hybrid protein represents a readily obtainable source of biologically active (STb) enterotoxin that may prove useful in studies to determine the mode of toxin action.

Infect Immun, 1990 Nov, 58(11), 3613 - 20
Genetic relationships among strains of avian Escherichia coli associated with swollen-head syndrome; White DG et al.; Genetic diversity among 22 Escherichia coli strains isolated from chickens with swollen-head syndrome (SHS), an acute respiratory disease of domestic poultry, and 93 strains isolated from birds with colibacillosis was assessed on the basis of allelic variation at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis . SHS isolates from Spain and Canada were polymorphic at 14 loci and were classified into 19 multilocus genotypes, defining clones that differed on average at 34% of the loci . In most cases, SHS isolates of different clonal genotypes were distinct in O:H serotype and expressed different fimbrial antigens . Comparisons with 93 isolates obtained from birds with colibacillosis revealed enzyme polymorphisms at 17 of 20 loci, with an average of 3.5 alleles per locus . In the total sample, 56 clonal genotypes were distinguished, with 27 (23%) of the isolates belonging to one of three common clones . Both SHS and colibacillosis isolates were genetically diverse, with an average single-locus diversity of 0.36, indicating that a wide variety of naturally occurring bacterial clones is associated with these acute avian infections . Six previously defined groups of clones identified in diseased birds from the United States were represented in isolates from Spain, indicating that similar clones occur in widely separated geographic areas . In addition, one group of SHS isolates was closely related to a recognized widespread clone complex incriminated in human septicemia and meningitis . The results suggest that certain strains implicated in SHS infections belong to a clone complex whose members have special attributes that promote involvement in invasive diseases in humans and animals.

Hepatology, 1990 Nov, 12(5), 1125 - 8
Immunization of woodchucks with recombinant hepatitis delta antigen does not protect against hepatitis delta virus infection; Karayiannis P et al.; To assess the role of immunization against hepatitis delta antigen in the prevention of hepatitis delta virus infection, woodchuck carriers of woodchuck hepatitis virus were immunized with a 64 amino acid portion of hepatitis delta antigen from its N-terminal region . The protein was expressed in Escherichia coli and contained a major immunogenic epitope . A significant anti-hepatitis delta response was observed that did not, however, protect the animals from hepatitis delta virus superinfection . Unexpectedly, the period of detectable viremia was longer in the immunized than in the control animals . We conclude that immunization with this recombinant hepatitis delta antigen does not afford protection against subsequent hepatitis delta virus exposure.

Virology, 1990 Nov, 179(1), 16 - 25
Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus; Manning DS et al.; A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish . When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells . When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized . In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor . Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells . Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame . A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria . The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.

J Immunol, 1990 Nov 1, 145(9), 3033 - 40
Endothelial and leukocyte forms of IL-8 . Conversion by thrombin and interactions with neutrophils; Hebert CA et al.; We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers . IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis . The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called {ser-IL-8}72) and the other 77 amino acids (an N-terminal extended form herein called {ala-IL-8}77) . IL-8 derived from T lymphocytes and monocytes is predominantly {ser-IL-8}72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in {ala-IL-8}77 . We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli . Thrombin was found to efficiently convert {ala-IL-8}77 to {ser-IL-8}72 . In contrast, urokinase and tissue-type plasminogen activator were unable to cleave {ala-IL-8}77, and trypsin generated multiple IL-8 cleavage fragments . In competitive binding assays using 125I{ala-IL-8}77 neutrophils exhibited a twofold preference for {ser-IL-8}72 over {ala-IL-8}77 . Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90% . However, {ser-IL-8}72 was approximately 10-fold more potent than {ala-IL-8}77 in these assays (ED50 approximately 0.3 nM for {ser-IL-8}72 vs approximately 3 nM for {ala-IL-8}77 . Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils {{ser-IL-8}72 (ED50 greater than 10 nM) was two- to three-fold more potent than {ala-IL-8}77}, although in this regard they were less active than FMLP . Our data suggest that {ala-IL-8}77 and {ser-IL-8}72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the {ser-IL-8}72 form . In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.

EMBO J, 1990 Nov, 9(11), 3761 - 5
Periodic formation of the oriC complex of Escherichia coli; Gayama S et al.; We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe . In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event . Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication . Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature . Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture . However, the second periodic peak did not appear when rifampicin or chloramphenicol was added . Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature . We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.

Mol Endocrinol, 1990 Nov, 4(11), 1689 - 97
Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice; Hammer GD et al.; All aspects of POMC biosynthesis exhibit tissue-specific regulation . The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus . POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine . To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes . The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E . coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene . Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry . In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs . Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts . There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment . X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Scand J Gastroenterol, 1990 Nov, 25(11), 1129 - 36
Influence of individual bile acids in Escherichia coli peritonitis; Andersson R et al.; Previous studies have shown that intraperitoneal bile increases bacterial growth and mortality in Escherichia coli peritonitis in the rat . The purpose of the present study was to determine a) the influence of bile acids (cholic, deoxycholic, or chenodeoxycholic) and bilirubin on survival, bacterial growth, and superoxide release by peritoneal phagocytes in this model, and b) the effect of bile acids on bacterial growth and endotoxin release when incubated with E . coli in vitro . Each of the bile acids aggravated the E . coli peritonitis, with increased bacterial counts in the peritoneal cavity and in blood and increased mortality . Deoxycholic acid was the most deleterious of the bile acids, causing suppression of superoxide release by peritoneal phagocytes, like whole bile . In vitro, bile acids did not seem to affect growth of E . coli, but cholic and deoxycholic acid seemed to enhance the release of endotoxin . It is concluded that the bile acids are responsible for the noxious effect of bile in E . coli peritonitis . It is suggested that the detergent properties of bile acids aggravate the peritonitis by solubilizing the cell membranes of both bacteria and phagocytes.

J Pediatr Surg, 1990 Nov, 25(11), 1183 - 4
Multiple small bowel perforations in leukemia secondary to Epstein-Barr virus lymphoma with survival: a case report; Di Lorenzo M et al.; This is the case report of a 4-year-old white boy who was diagnosed as having acute lymphoblastic leukemia (ALL) in November 1985 . While in remission and on maintenance chemotherapy, he developed a primary Epstein-Barr virus (EBV) respiratory infection in October 1986 . On October 27, 1986 a plain abdominal radiograph taken for abdominal distention showed free air . At celiotomy, multiple nodules were noted to stud the small bowel . Central necrosis of these nodules with perforations were present in the distal small bowel . Resections and end-to-end anastomoses were performed . Three days later the patient again had a similar acute abdominal episode . At reexploration, similar lesions in the liver, kidney, duodenum, proximal jejunum, and colon were found . Liver biopsy as well as intestinal resections and end-to-end anastomoses were performed, along with a loop ileostomy . Polymorphic B-cell lymphoma positive for EBV was found in the specimens . After cessation of chemotherapy and institution of abdominal radiotherapy, the hepatic and renal lesions were seen to resolve on computed tomography scan . The patient's course was complicated by the development of cervical and mediastinal abscesses that were drained, and E coli sepsis accompanied by chronic diarrhea requiring intravenous hyperalimentation . By January 1988, he appeared to be recovering . His ileostomy was closed in March 1988 . Despite cessation of chemotherapy since October 1986, the patient is now well and in complete remission.

Appl Environ Microbiol, 1990 Nov, 56(11), 3612 - 4
Does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?
Koizumi J, Zhang M, Imanaka T, Aiba S.
Thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated Sephadex G-200 particles . Lys in place of Gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8965 - 9
Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the {2Fe-2S} cluster; Werth MT et al.; Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers . In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, {2Fe-2S}; center 2, {4Fe-4S}; center 3, {3Fe-4S}) have been assembled . The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this {2Fe-2S} center is essential for optimal enzymatic activity . The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(III) site of the reduced cluster . The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8950 - 4
An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology; Geller AI et al.; We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells . pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain . HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors . A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type . In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors . We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A . pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia . Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

Arch Biochem Biophys, 1990 Nov 1, 282(2), 372 - 6
pH probes respond to redox changes in cytochrome o; Sedgwick EG et al.; N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids . We have shown (Sedgwick, E . G., and Bragg, P.D . (1988) FEBS Lett . 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles . We now show that the pH probes pyranine and quinacrine behave similarly to NPN . The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium . It is insensitive to uncouplers . The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid . The NPN response requires added phospholipid . Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome . This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.

Am Surg, 1990 Nov, 56(11), 707 - 14
Endotoxin and calcium effect on metabolism; Kasden SE et al.; The effects of calcium and endotoxin were investigated in vivo and in vitro using the A/J, retired white male breeder mouse model . Intravenous administration of increasing calcium doses (3.75, 4.37, and 4.75 mg Ca/g-BW) resulted in increasing serum calcium levels (11.4, 14.4, 15.2, respectively vs . animals not injected, 9.5 mg/dl) . Mice injected with E . coli bacterial endotoxin after injection of calcium demonstrated significantly (P less than 0.05) lowered serum calcium concentrations (10.5, 12.1, 12.5, respectively) . The magnitude of serum calcium reduction was related to the level just prior to endotoxin administration . Mitochondria were incubated using a Percoll purification step . The effect of pretreating the animal in vivo with calcium, endotoxin or calcium plus endotoxin versus control was investigated in both liver and skeletal muscle . When mitochondria were incubated in medium containing EGTA (free Ca less than 1 X 10(-9)) . no significant difference between groups was observed in either (RCR) or mitochondrial calcium content . When mitochondria were incubated in EGTA buffers containing calcium (free Ca approximately 7 X 10(-7) liver, 1 X 10(-7) muscle) significant differences in RCR and mitochondrial calcium content were observed . Liver RCR increased significantly with calcium or endotoxin pretreatment (2.20, 2.43 vs . 1.86) . Muscle mitochondrial RCR significantly decreased with calcium administration (5.02 vs . 6.79) . Both liver and muscle mitochondrial calcium content significantly decreased (2.54 vs . 27.32, and 6.46 vs . 25.82 nMole Ca/mg mitochondrial protein) . Preloading with calcium prevented the decline in mitochondrial calcium content . Findings suggest that the reduction in intramitochondrial calcium content during endotoxemia could be an effector of metabolic derangement specifically through calcium-dependent intramitochondrial enzymes.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8587 - 91
Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene; Itaya M; An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12 . By screening a library of E . coli DNA for clones that suppressed RNase H deficiency of an E . coli rnh mutant, a clone was obtained that produced a protein with RNase H activity . The overexpressed RNase HIII protein in E . coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate . The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence . The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E . coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity . The presence of a second RNase H in E . coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8373 - 7
Superhelical torsion in cellular DNA responds directly to environmental and genetic factors; McClellan JA et al.; Superhelical tension of DNA in living bacteria is believed to be partially constrained by interaction with proteins . Yet DNA topology is a significant factor in a number of genetic functions and is apparently affected by both genetic and environmental influences . We have employed a technique that allows us to estimate the level of unconstrained superhelical tension inside the cell . We study the formation of cruciform structures by alternating adenine-thymine sequences in plasmid DNA by in situ chemical probing . This structural transition is driven by superhelical torsion in the DNA and thus reports directly on the level of such tension in the cellular DNA . We observe that the effect of osmotic shock is an elevation of superhelical tension; quantitative comparison with changes in plasmid linking number indicates that the alteration in DNA topology is all unconstrained . We also show that the synthesis of defective topoisomerase leads to increased superhelical tension in plasmid DNA . These experiments demonstrate that the effect of environmental and genetic influences is felt directly at the level of torsional stress in the cellular DNA.

Mol Cell Biol, 1990 Nov, 10(11), 5700 - 6
Heterogeneous expression of poliovirus receptor-related proteins in human cells and tissues; Freistadt MS et al.; Portions of the cellular receptor for poliovirus were expressed in Escherichia coli as fusion proteins with the product of the trpE gene . One of two antireceptor antisera obtained by immunizing rabbits with the fusion proteins blocked poliovirus infection . Western immunoblot analyses demonstrated that poliovirus receptor-related proteins were expressed in HeLa cells and a variety of human tissues, including those that are not sites of poliovirus replication . Tissue-specific variation in electrophoretic mobility, immunoreactivity, and subunit arrangement of poliovirus receptor-related proteins was observed . These results demonstrate that poliovirus tissue tropism cannot be explained by a limited distribution of receptor polypeptide, but may be the result of alternative splicing, posttranslational modifications, or both . In addition, the widespread but heterogeneous expression of the receptor suggests that the protein may have an important endogenous function.

J Bacteriol, 1990 Nov, 172(11), 6403 - 10
EntG activity of Escherichia coli enterobactin synthetase; Staab JF et al.; The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G) . However, sequencing data showed that there is no separate entG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified . In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity . Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG- . PJS43 had a Tn5 inserted 198 bp from the entB termination codon, and pJS100 had the last 25 codons of entB deleted . Plasmids isolated with Tn5 insertions in the 5' half of entB had the phenotype EntB- EntG+ . These latter Tn5 mutations were EntB- EntG- when moved to the bacterial chromosome . Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots . Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect . Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized . Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB . In summary, EntG activity was found to be encoded by the entB 3' terminus . The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.

J Bacteriol, 1990 Nov, 172(11), 6333 - 8
Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product; Cotter PA et al.; The aerobic respiratory chain of Escherichia coli contains two terminal oxidases that catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water . They are the cytochrome o oxidase complex encoded by cyoABCDE and the cytochrome d oxidase complex encoded by cydAB . To determine how these genes are regulated in response to a variety of environmental stimuli, including oxygen, we examined their expression by using lacZ protein fusions in wild-type and fnr mutant strains of E . coli . Anaerobic growth resulted in a 140-fold repression of cyoA'-'lacZ expression relative to aerobic growth and a 3-fold increase in cydA'-'lacZ expression . Anaerobic repression of both fusions was mediated in part by the fnr gene product, as evidenced by a 30-fold derepression of cyoA'-'lacZ expression and a 4-fold derepression of cydA'-'lacZ expression in an fnr deletion strain . Supplying wild-type fnr in trans restored wild-type repression for both fusions . Fnr thus functions as an anaerobic repressor of both cyoABCDE and cydAB expression . Reduced-minus-oxidized difference spectrum analyses of cell membranes confirmed the effect of the fnr gene product on the production of cytochrome d oxidase in the cell . Based on the pattern of anaerobic cydAB expression observed, we propose the existence of a second, as yet unidentified, regulatory element that must function either to activate cydAB expression as oxygen becomes limiting or to repress cydAB expression aerobically . Whereas cytochrome o oxidase encoded by cyoABCDE appears to be produced only under oxygen-rich growth conditions, in keeping with its biochemical properties, cytochrome d oxidase is expressed moderately aerobically and is elevated yet further when oxygen becomes limiting so that the organism can cope better under oxygen starvation conditions . We also examined cyoABCDE and cydAB expression in response to growth on alternative carbon compounds and to changes in the culture medium pH and osmolarity.

J Bacteriol, 1990 Nov, 172(11), 6194 - 203
Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination; Gerlitz M et al.; The broad-host-range plasmid RP4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb PstI C fragment . The essential functions were assigned to a region of 2.2 kb between fiwA and IS21 (IS8) . On the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kDa were identified . Evidence for the expression of another polypeptide was found . A putative divergent promoter was localized in an intergenic region and is suggested to be responsible for transcription of these genes . It was found that the RP4 par region includes a function resolving plasmid dimers . The 24-kDa polypeptide is considered to function as a resolvase, since its predicted amino acid sequence shows homology to sequences of resolvases of the Tn3 family . Furthermore, palindromes present in the intergenic region containing the divergent promoter resemble repeat structures specific for res sites of Tn3-related transposons . However, it was found that dimer resolution itself was not sufficient for stabilization; additional functions, including the other two polypeptides, seemed to play an important role . These results suggested that RP4 contains a complex stabilization system involving resolution of plasmid dimers during cell division, thus ensuring the delivery of at least one copy to each daughter cell.

New Biol, 1990 Nov, 2(11), 975 - 91
Action of an RNA site at a distance: role of the nut genetic signal in transcription antitermination by phage-lambda N gene product; Whalen WA et al.; The N gene product of Escherichia coli phage lambda is a transcriptional activator that captures the host RNA polymerase and modifies it to a termination-resistant form, permitting gene expression in two large polycistronic operons of the phage genome . Antitermination in vitro requires at least one host factor called NusA, which directly binds the N protein as well as RNA polymerase, and also a transcribed cis-acting site known as nut, within which lies the hypothesized N-recognition signal, boxB . BoxB is an interrupted palindrome capable of forming a hairpin in the mRNA . Inhibition studies with complementary DNA oligonucleotides provide evidence for a direct role of the boxB hairpin in antitermination . Kinetic studies of transcript elongation reveal that the boxB hairpin does not induce an appreciable pause to hold polymerase captive for engagement by N and NusA . Moreover, the efficiency of antitermination remains virtually the same whether N and NusA are added early, prior to nut site transcription, or added later, after the polymerase has already transcribed past the nut site . After transcription of the nut site, RNA polymerase remains susceptible to modification by N and NusA for an appreciable amount of time and distance, and the nut site DNA becomes dispensable for this modification . These results lead to the hypothesis that the boxB RNA hairpin acts in a manner analogous to the DNA enhancers, binding N and mediating a productive polymerase-NusA-N interaction by mRNA looping.

Mol Biol (Mosk), 1990 Nov-Dec, 24(6), 1631 - 9
{Expression of gene coding for the NS1 protein of the tick-borne encephalitis virus in Escherichia coli cell}; Pugachev KV et al.; Two possible forms of tick-borne encephalitis virus (TBE) gene NS1 (called NS1' and NS1) were constructed using two overlapping cDNA-fragments of TBE genome and synthetic DNA fragments . This genes were expressed in E . coli cells in expression vector pUR290 as individual proteins or fusion with bacterial beta-galactosidase . The proteins NS1 (Mw . 39 kDa), beta-galactosidase-NS1' (Mw . 162 kDa) and beta-galactosidase-NS1 (Mw . 155 kDa) were effectively synthesized under the Plgc-promoter induction conditions . Expression of NS1' gene results in the formation of two virus-specific proteins (Mw . 46 and 44 kDa) . All bacterial analogs of NS1 protein fixed monoclonal and polyclonal antibodies specific to viral NS1.

Mol Microbiol, 1990 Nov, 4(11), 1941 - 6
The Escherichia coli unc transcription terminator enhances expression of uncC, encoding the epsilon subunit of F1-ATPase, from plasmids by stabilizing the transcript; Patel AM et al.; The effect of the unc transcription terminator on expression of uncC, encoding the epsilon subunit of Escherichia coli F1-ATPase, from plasmids was studied . The cloned sequence in pTK1 included the uncC ribosome binding site, the uncC structural gene, and the unc transcription terminator . The cloned region in pSD37 was similar, but lacked the unc transcription terminator . Transformants carrying pTK1 produced the epsilon subunit of F1-ATPase, encoded by uncC, in 10-fold greater abundance than transformants carrying pSD37 . Northern blots revealed similar differences in the steady-state uncC mRNA levels . The half-life of the message transcribed from pTK1 was 90-100 seconds, while that from pSD37 was 25-30 seconds . These studies indicate the importance of message stabilization through features at the 3' end of the transcript in ensuring adequate production of epsilon . We have exploited this stabilization to develop a simple, efficient, and gentle method of purifying the overproduced epsilon subunit.

Mol Microbiol, 1990 Nov, 4(11), 1847 - 52
Site-directed mutagenesis of the 'zinc finger' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora; Fu YH et al.; The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation . The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain . The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity . The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene . A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity . The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8476 - 80
Genetic recombination in Escherichia coli: Holliday junctions made by RecA protein are resolved by fractionated cell-free extracts; Connolly B et al.; Escherichia coli RecA protein catalyzes reciprocal strand-exchange reactions between duplex DNA molecules, provided that one contains a single-stranded gap or tail, to form recombination intermediates containing Holliday junctions . Recombination reactions are thought to occur within helical RecA-nucleoprotein filaments in which DNA molecules are interwound . Structures generated in vitro by RecA protein have been used to detect an activity from fractionated E . coli extracts that resolves the intermediates into heteroduplex recombinant products . Resolution occurs by specific endonucleolytic cleavage at the Holliday junction . The products of cleavage are characteristic of patch and splice recombinants.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8227 - 31
Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery; Oliver DB et al.; Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E . coli mutants isolated . Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E . coli plasma membrane . We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition . Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition . These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.

J Bacteriol, 1990 Nov, 172(11), 6607 - 10
Mutations conferring resistance to azide in Escherichia coli occur primarily in the secA gene; Fortin Y et al.; Mutant strains of Escherichia coli were screened for the ability to grow on L agar plates containing 3.4 or 4.6 mM sodium azide . Most mutants had mutations located in the leucine region, presumably at the azi locus . Two of these mutants were found to have a mutation in the secA gene, but expression of the resistance phenotype also required the presence of upstream gene X . While a plasmid carrying the X-secA mutant gene pair was able to confer azide resistance to a sensitive host, a similar plasmid harboring the wild-type secA allele rendered a resistant strain sensitive to azide, indicating codominance of the two alleles . That azide inhibits SecA is consistent with the fact that SecA has ATPase activity, an activity that is often prone to inhibition by azide.

Mol Microbiol, 1990 Nov, 4(11), 1831 - 8
Interconversion of the DNA-binding specificities of two related transcription regulators, CRP and FNR; Spiro S et al.; In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA-binding domains containing analogous helix-turn-helix motifs . The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA-recognition helices . In FNR, Glu-209 and Ser-212 are essential residues for the recognition of FNR sites . A V208R substitution confers CRP-site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth . In contrast, changes at two (V208R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP-site binding specificity . In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR . Altering Asp-199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP . Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.

Neuroreport, 1990 Nov-Dec, 1(3-4), 203 - 6
CNTF rescues motoneurons from ontogenetic cell death in-vivo, but not in-vitro; Wewetzer K et al.; We studied the effect of CNTF (ciliary neurotrophic factor, human recombinant and chick) on the survival of motoneurons in the embryonic chick lumbar spinal cord during the period of ontogenetic cell death . Daily applications of 5 micrograms CNTF to the chorionic-allantoic membrane from embryonic day 6 (E6) to E9 maintained approximately 15,500 motoneurons as opposed to 13,200 in controls . In contrast, CNTF failed to promote the survival of cells in spinal cord cultures enriched for motoneurons . These results suggest that CNTF may regulate motoneuron survival in-vivo, but its mode of action remains to be elucidated.

Plant Mol Biol, 1990 Nov, 15(5), 723 - 33
Cloning of salinity stress-induced genes from the salt-tolerant nitrogen-fixing cyanobacterium Anabaena torulosa; Apte SK et al.; A subtractive hybridization procedure was used to clone genes of the cyanobacterium Anabaena torulosa expressed in response to salt stress . The method uses total RNA from salt-treated cells, labeled in vitro, as the probe . Hybridization to restriction digests of total DNA was used for interspecies comparison; the procedure was also successful for isolation of cosmids by colony hybridization, semiquantitative dot blots, and cosmid characterization by Southern blotting . Analysis of eleven independent cosmids containing genes whose transcription is abundantly induced by salt suggests that a substantial portion of the A . torulosa genome, probably in excess of 100 kilobases, responds to salt.

J Neurosci Res, 1990 Nov, 27(3), 427 - 39
Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line; Short MP et al.; Retrovirus vectors only integrate into the genome of dividing cells and can thus be used to selectively infect tumor cells in the adult rat brain . Gene delivery was assessed by using the retrovirus BAG vector, which bears the Escherichia coli lacZ gene under the MoMLV LTR promoter-enhancer element, and by histochemical staining for bacterial beta-galactosidase activity . Direct injection of this vector (90-900 cfu) into the adult rat brain, with or without prior inoculation of C6 glioma cells (2 x 10(5) cells) resulted in labeling of only a few cells as assessed 1 week later . When the psi 2-BAG packaging line was grafted into the brain, labeled psi 2-BAG cells could be found after 1 day, but not after 5 days, following grafting, suggesting that the grafted cells had been rejected and that no endogenous cells had integrated released vector, or that expression of lacZ had been turned off . In contrast, when the psi 2-BAG packaging line was grafted into a brain region, which had been inoculated previously with rat C6 glioma cells (2 x 10(5) cells), beta-galactosidase labeling of these tumor cells, identified by immunocytochemistry for glial fibrillary acidic protein and S100, could be demonstrated 10 days later . Thus, grafting of retrovirus packaging lines into adult brain provides a mean to infect tumor cells in situ . The grafted packaging cells may continue to release retrovirus particles for an extended period, thus infecting more cells at the stage of division appropriate for viral integration, as compared to inoculation of the virus alone . Grafting of retrovirus packaging cell lines could be used to selectively deliver "killer" or "suppressor" genes to tumor cells in the brain to curtail their growth.

J Photochem Photobiol B, 1990 Nov, 7(2-4), 317 - 36
Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin; Trabalzini L et al.; The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology . The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli . The photoadduct distribution of psoralen is also studied for comparison . Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine . Alternating (A-T)n sequences are hot spots for visnagin photoaddition . This is a property shared with furocoumarins . TTT sites are also quite reactive to visnagin, as they are to methylated angelicins . In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well . Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly . The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al . The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al . The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts . The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.

Mol Gen Genet, 1990 Nov, 224(2), 222 - 31
The cyanelle S10 spc ribosomal protein gene operon from Cyanophora paradoxa; Michalowski CB et al.; In Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial . Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons . The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria . Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5'-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18- rps5-3' . Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster . The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs . These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of approximately 7500 nucleotides . The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments . Transcription terminates 3' of rps5 where a 40 bp hairpin with one mismatch (-42.2 kcal) may be folded . Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript . A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.

Circ Shock, 1990 Nov, 32(3), 231 - 42
Therapeutic intervention in a rat model of ARDS: I . Dual inhibition of arachidonic acid metabolism; Turner CR et al.; The protective effects of altered arachidonic acid metabolism, using either methylprednisolone or a dual cyclooxygenase and 5-lipoxygenase inhibitor (SK&F 86002), were compared in a rat model of adult respiratory distress syndrome (ARDS) . Rats were either unexposed (n = 9) or pretreated with vehicle (n = 25), 100 mg/kg SK&F 86002 (n = 8) or 30 mg/kg methylprednisolone (MP, n = 7) 1 h prior to the intratracheal administration of 7 mg/kg aerosolized endotoxin (LPS) or phosphate buffered saline (PBS) . Twenty-four hours later, blood samples were collected and the rats were anesthetized and exsanguinated . The lungs were surgically removed, weighed and bronchoalveolar lavage (BAL) was performed . LPS caused 30-35% mortality and induced significant differences in body weight, BAL erythrocyte and neutrophil counts, lung wet/dry weight ratio (W/D), total BAL protein (TP), hemoglobin (Hb), hematocrit (HCT), and circulating leukocyte and platelet counts as compared with controls . Pretreatment with MP reduced mortality to zero and also attenuated the LPS-induced alterations in body weight, W/D, TP, BAL erythrocyte count, and circulating platelet count . However, MP exacerbated LPS-induced increases in Hb, HCT and circulating neutrophil counts while enhancing lymphopenia . Pretreatment with SK&F 86002 also reduced mortality to zero and attenuated LPS-induced alterations in W/D, TP, HCT and circulating platelet count . Like MP, SK&F 86002 exacerbated the LPS-induced lymphopenia, and increased circulating neutrophils above baseline values . We conclude that both MP and SK&F 86002 provided protection against LPS-induced responses in this model of ARDS . Mechanistically, this indicates the critical role of eicosanoid mediators in this model . Therapeutically, SK&F 86002, or a similar compound, may be beneficial in preventing the acute phase responses so harmful to ARDS patients.

J Bacteriol, 1990 Nov, 172(11), 6518 - 28
Metabolic relationships between pyridoxine (vitamin B6) and serine biosynthesis in Escherichia coli K-12; Lam HM et al.; We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5,5', and 6 of the pyridoxine ring . This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicted on the homology between PdxB and SerA, the structural similarity between serine and 4-hydroxythreonine, and the possible involvement of SerC in pyridoxine biosynthesis . Several predictions of this hypothetical scheme were tested . Consistent with the proposed pathway, supplement inhibition patterns strongly suggest that SerA enzyme acts in a an alternate pathway of pyridoxine biosynthesis in pdxB mutants . Direct enzyme assays detected erythrose-4-phosphate dehydrogenase activity in crude extracts, which again supports the proposed pathway . Chromosomal insertions in serC caused a requirement for pyridoxine, serine, and aromatic compounds, which directly verified that SerC functions in the pyridoxine biosynthetic pathway . Complementation analysis showed that pdxF and pdxC mutations reported previously are most likely alleles of serC . Growth of serC chromosomal insertion mutants on glycoalaldehyde was found to occur without acquisition of second-site mutations and confirmed that pdxB and serC, but not pdxA, function in the same branch of the pyridoxine pathway . In addition, serC::mini-Mu d insertions revealed that the complex serC-aroA operon lacks internal promoters, that the amino terminus of SerC is not strictly essential for activity, and that antisense transcription occurs in the serC-aroA operon . Growth responses of pdxA, pdxB, and serC mutants to beta-hydroxypyruvate, D-alanine, and glycolate could also be reconciled with the proposed pathway . Finally, the proposed scheme is consistent with previous isotope labeling data and accounts for several other observations about pyridoxine biosynthesis . Together, these physiological and biochemical analyses support the proposed pathway and an evolutionary scenario in which this branch of the pyridoxine pathway evolved from the serine pathway by gene recruitment.

J Bacteriol, 1990 Nov, 172(11), 6363 - 71
Expression of the Synechocystis sp . strain PCC 6803 tRNA(Glu) gene provides tRNA for protein and chlorophyll biosynthesis; O'Neill GP et al.; In the cyanobacterium Synechocystis sp . strain PCC 6803 (Synechocystis 6803) delta-aminolevulinic acid (ALA), the sole precursor for the synthesis of the porphyrin rings of heme and chlorophyll, is formed from glutamate activated by acylation to tRNA(Glu) (G . P . O'Neill, D . M . Peterson, A . Schon, M . W . Chen, and D . Soll, J . Bacteriol . 170:3810-3816, 1988; S . Rieble and S . I . Beale, J . Biol . Chem . 263:8864-8871, 1988) . We report here that Synechocystis 6803 possesses a single tRNA(Glu) gene which was transcribed as monomeric precursor tRNA and matured into the two tRNA(Glu) species . They differed in the extent of modification of the first anticodon base, 5-methylaminomethyl-2-thiouridine (O'Neill et al., 1988) . The two tRNA species had equivalent capacities to stimulate the tRNA-dependent formation of ALA in Synechocystis 6803 and to provide glutamate for protein biosynthesis in an Escherichia coli-derived translation system . These results are in support of a dual role of tRNA(Glu) . The levels of tRNA(Glu) were examined by Northern (RNA) blot analysis of cellular RNA and by aminoacylation assays in cultures of Synechocystis 6803 in which the amount of chlorophyll synthesized was modulated over a 10-fold range by various illumination regimens or by the addition of inhibitors of chlorophyll and ALA biosynthesis . In these cultures, the level of tRNA(Glu) was always a constant fraction of the total tRNA population, suggesting that tRNA(Glu) and chlorophyll levels are regulated independently . In addition, the tRNA(Glu) was always fully aminoacylated in vivo.

Infect Immun, 1990 Nov, 58(11), 3796 - 801
Effects of Escherichia coli hemolysin on endothelial cell function; Suttorp N et al.; Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E . coli infections . The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E . coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml) . Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner . The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function . The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation . In addition, E . coli hemolysin stimulated prostacyclin synthesis in endothelial cells . This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+ . An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes . These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes . We conclude that endothelial cells are highly susceptible to E . coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin.

Pharmacol Toxicol, 1990 Nov, 67(5), 387 - 91
Antidotal effect of lipopolysaccharide against acetaminophen-induced mortality in mice; Ishikawa M et al.; The possibility that pretreatment with LPS (lipopolysaccharide obtained from Escherichia coli), an immune system stimulant and interferon inducer, could prevent the hepatotoxic effects of acetaminophen (NAPAP) was investigated in mice . When mice were pretreated with LPS (4 mg/kg), intraperitoneally for 24 hr the mortality caused by NAPAP was considerably reduced . Histological examination of the livers and leakage of the enzymes into the blood demonstrated that NAPAP-induced necrosis was decreased in LPS-treated mice compared to that induced by NAPAP alone . Pretreatment with 400 mg/kg or 800 mg/kg of NAPAP decreased the amount of covalently-bound acetaminophen metabolites . Since the level of hepatic glutathione and microsomal cytochrome P-450 were depressed in these experiments, it is concluded that LPS depresses the cytochrome P-450 species responsible for the formation of the toxic metabolites and that less reactive species are available for binding to cell macromolecules.

Res Microbiol, 1990 Nov-Dec, 141(9), 1117 - 29
A human DNA telomeric fragment contains yeast ARS and mitotic stabilizing sequences; Ascenzioni F et al.; A minilibrary of human DNA fragments was prepared in the vector YIP5 from a DNA preparation enriched for telomeric sequences . Screening of the library produced one clone that hybridized to the TTAGGG sequence . The cloned DNA fragment was shown to be telomeric by a number of criteria . In situ hybridization to metaphase human chromosomes showed that the fragment hybridized to the tips of all human chromosomes . The fragment contained at least two yeast autonomously replicating sequences (ARS) and stabilizing sequences, since it transformed Saccharomyces cerevisiae with high efficiency, giving rise to clones which were mitotically stable under non-selective growth.

J Biochem Biophys Methods, 1990 Nov-Dec, 21(4), 289 - 97
A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli; Mehrpouyan M et al.; A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells . Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme . The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.

Res Virol, 1990 Nov-Dec, 141(6), 597 - 610
Expression vectors for quantitating in vivo translational ambiguity: their potential use to analyse frameshifting at the HIV gag-pol junction; Cassan M et al.; Translational errors are necessary so as to allow gene expression in various organisms . In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting . Here we present an experimental system that permits quantification of translational errors in vivo . It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter . Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthrough or frameshift, respectively, to give rise to an active product . This system is sensitive enough to detect background errors in mammalian cells . In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest . This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV . The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells . The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect . This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.

Biochimie, 1990 Nov, 72(11), 835 - 43
Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12; Yancey SD et al.; Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 {1} . In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene . In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C . The pnp-200 mutation alone does not affect cell growth or mRNA stability . A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.

Biochimie, 1990 Nov, 72(11), 825 - 34
RNase III cleavages in non-coding leaders of Escherichia coli transcripts control mRNA stability and genetic expression; Regnier P et al.; The primary transcripts of the rpsO-pnp, rnc-era-recO and metY-nusA-infB operons of E coli are each processed by RNase III, upstream of the first translated gene, in hair-pin structures formed by the 5' non-coding leader . The mRNAs of the 3 operons, of which the 5' terminal motifs have been removed by RNase III, decay significantly more rapidly than the uncut transcripts which accumulate in the RNase III deficient strain . The rapid decay of a primary transcript of the metY-nusA-infB operon, initiated at a secondary promoter in the vicinity of the RNase III sites, suggests that the 5' features upstream of the RNase III cutting sites are responsible for the stability of the uncut RNAs . RNase III autocontrols its own expression by removing the 5' motif which stabilizes its mRNA . Similarly, the synthesis of polynucleotide phosphorylase and of protein Era are also controlled by RNase III cleavages which trigger the degradation of their messengers . The role of RNase III in the regulation of gene expression and the possible mechanisms of mRNA stabilization and of 5' to 3' decay initiated by RNase III processing are discussed.

Biochimie, 1990 Nov, 72(11), 803 - 11
Phage fl mRNA processing in Escherichia coli: search for the upstream products of endonuclease cleavage, requirement for the product of the altered mRNA stability (ams) locus; Kokoska RJ et al.; In Escherichia coli infected with the filamentous phage f1, a number of the polycistronic phage mRNA species are generated through post-transcriptional processing by host nuclease activity . In this paper we review experimental evidence assessing whether known RNases are involved in mediating these processing events, and we use S1 nuclease mapping methods to visualize putative upstream products of endonuclease cleavage . By examining f1 processing in a phage-infected host bearing a temperature-sensitive allele of the altered message stability locus (ams), we show that production of the major processed species requires a component of the host cell which functions in the messenger RNA decay process.

Agents Actions, 1990 Nov, 31(3-4), 341 - 4
Prostaglandin F2 alpha stimulates gluconeogenesis in the perfused rat liver and this effect is blunted in livers from endotoxin infused rats; Spitzer JA et al.; Prostaglandin F2 alpha stimulates gluconeogenesis from lactate plus pyruvate in perfused rat livers . Continuous endotoxin infusion for 30 h in vivo prior to F2 alpha stimulation blunts this effect.

Agents Actions, 1990 Nov, 31(3-4), 275 - 9
Ambroxol inhibits interleukin 1 and tumor necrosis factor production in human mononuclear cells; Bianchi M et al.; We have studied the effect of Ambroxol on the production of Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) in human mononuclear cells (MNC) . For this purpose MNC were cultured for 24 h in the presence of endotoxin and different doses of Ambroxol . The results indicate that Ambroxol markedly inhibited IL-1 and TNF production at doses of 10-100 microgram ml, without any apparent toxicity.

Agents Actions, 1990 Nov, 31(3-4), 263 - 74
An assessment of plasma histamine concentrations during documented endotoxic shock; Brackett DJ et al.; Recent reviews of the literature involving histamine release during sepsis and endotoxemia have reported that the majority of the studies are inconclusive due to inadequate assays or experimental protocols . In a controlled experimental setting we have employed a specific and sensitive radioenzymatic assay to determine plasma histamine concentrations temporally during documented endotoxin-induced shock in the conscious rat . Cardiovascular and metabolic measurements for the control group (n = 7) were normal during the study period . Endotoxin (n = 8, LD/90-24 hrs.) induced an early transient hypotensive episode and increase in systemic vascular resistance and a sustained decrease in cardiac index and central venous pressure and increase in heart and respiratory rates . Hypoglycemia and hyperlacticemia were present at the end of the four-hour study period . The small intestine was severely hemorrhaged in all animals given endotoxin . Histamine concentrations for the control group were unchanged throughout the study period . Contrary to previous reports, this model of endotoxemia revealed unchanging histamine concentrations during the first 30 minutes which were temporally coincident with the characteristic early hypotensive episode evoked by endotoxin . The histamine concentrations at 60 and 240 minutes following endotoxin were increased two and three-fold, respectively, compared to the control group . Three of the 8 rats given endotoxin died before four hours; histamine concentrations in plasma taken when death appeared certain were 42, 91, and 174, compared to the control value of approximately 8 ng/ml . There was no clear association of the increases in plasma histamine with any of the parameters measured in this study: however, established histamine effects may have been masked by the pre-existing effects of other mediators known to be active during endotoxemia . In separate groups of animals endotoxin (n = 5) elicited early increases in plasma concentrations of norepinephrine (5-fold) and epinephrine (8-fold) that remained elevated for the 4-hour study period while the control group (n = 4) remained stable . This study establishes that a) plasma histamine concentrations are increased during endotoxemia, b) plasma histamine is not elevated during the initial hypotension episode following endotoxin, c) plasma histamine increases during the progression of endotoxic shock, and d) plasma histamine concentrations are extremely high prior to death.

Jpn Heart J, 1990 Nov, 31(6), 857 - 66
Diltiazem treatment in newborn canine endotoxic shock; Goto M et al.; The mortality of sepsis/septic shock continues to be high in newborns . However, there is no established method in its treatment . Although calcium channel blockers ameliorate the hemodynamic deterioration of adult circulatory shock, their effects on newborn endotoxic shock have not been elucidated . This study was performed in newborn dogs to investigate the effects of diltiazem on newborn endotoxic shock . Endotoxic shock was induced in newborn dogs (2-10 days old, 300-800 g) by an intravenous injection of E . coli lipopolysaccharide (LPS; 1.5 mg/kg), and diltiazem (DZ) at the dose of 300, 600 or 1200 micrograms/kg was administered intravenously 20 min prior to LPS injection . Hemodynamic changes were serially observed until 120 min after LPS injection . The heart rate, mean arterial pressure and cardiac output decreased after LPS injection, and systemic vascular resistance decreased . DZ at the dose of 600 micrograms/kg attenuated the decreases of MAP and cardiac output, but 300 and 1200 micrograms/kg of DZ exacerbated them . DZ at the dose of 1200 micrograms/kg decreased the heart rate, and DZ at all three doses attenuated the increase of systemic vascular resistance . Therefore, 600 micrograms/kg of DZ is beneficial in the treatment of endotoxic shock in newborn dogs.

Mol Microbiol, 1990 Nov, 4(11), 1993 - 2001
Identification and characterization of a gene and protein required for glycosylation in the yeast Golgi; Devlin C et al.; The MNN2 gene of Saccharomyces cerevisiae has been cloned by complementation of the mnn2 mutant phenotype scored by a change in cell surface carbohydrate structure resulting from a lack of alpha 1----2-mannose branching in the outer chain . The gene was subcloned as a 3 kb DNA fragment that integrated at the MNN2 locus, and a gene disruption yielded the mnn2 phenotype . A lacZ-MNN2 gene fusion protein, produced in Escherichia coli, was used to raise a specific antiserum that recognized a 65 kD wild-type yeast protein . This MNN2 gene product lacks N-linked carbohydrate but appears to be an integral membrane protein . Overproduction of MNN2p does not enhance the alpha 1----2-mannosyltransferase activity of yeast cells . The results suggest that MNN2p is a Golgi-associated protein that is involved in mannoprotein sorting rather than glycosylation.

Mol Microbiol, 1990 Nov, 4(11), 1911 - 9
Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis; Snapper SB et al.; Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria . While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA . Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors . This work describes the isolation and characterization of mutants of M . smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA . The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M . smegmatis chromosome, but seem to be specific for plasmid transformation . Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs . Efficient plasmid transformation of M . smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

Nippon Saikingaku Zasshi, 1990 Nov, 45(6), 921 - 9
{Flow cytometric analysis of oxidative burst of phagocytes with small amount of peripheral blood}; Shinomiya N et al.; We have developed a simple method for assessing the oxidative metabolic burst of peripheral blood leukocytes with a minute amount of whole peripheral blood by flow cytometry according to the method of Bass et al . with some modification . By this method, we can measure the H2O2 production by both granulocytes and monocytes in the same blood sample . The oxidative product formation by peripheral blood neutrophils can be monitored sequentially in the same mouse infected with E . coli . The mice infected intravenously with 0.1 LD50 of the bacteria showed increased basal activities from an early stage of infection; those infected intraperitoneally with the same dose of the bacteria showed a delayed enhancement . In case of infection with 0.01 LD50, the enhanced basal activities lasted for only a short period of time . The H2O2 production was correlated well with the clearance of the infected bacteria . These results demonstrated that the oxidative-product formation by peripheral blood neutrophils is affected by both the route and the dose of infection.

J Biochem (Tokyo), 1990 Nov, 108(5), 766 - 77
Structural characterization of non-acid glycosphingolipids in kidneys of single blood group O and A pigs; Holgersson J et al.; Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typed on their red blood cells as blood groups O and A . Glycolipid species were purified by HPLC and structurally characterized by thin-layer chromatography, mass spectrometry, proton NMR spectroscopy, degradation analysis, and reactivity with various monoclonal antibodies, Gal alpha 1-4Gal-specific E . coli bacteria, and lectins . Glucosyl-, globotriaosyl-, and globotetraosylceramides were the predominant molecular species with lactosyl- and globopentaosylceramides (IV3GalGb4Cer) as abundant constituents too . Small amounts of galactosyl- and digalactosylceramides were also present . In the blood group O pig kidneys, blood group H antigens based on four different core saccharides (types 1, 2, 4, and lactosyl core) were identified and the major blood group structure was V2FucIV3Gal-Gb4Cer . In the kidneys from the blood group A pig the corresponding blood group A antigens were found and in addition, a type 3 chain blood group A antigen was indicated by mass spectrometry and by its reactivity with a monoclonal antibody . Trace amounts of the type 2 chain-based X and Y antigens were found while blood group B antigens and the type 1 chain based Lewis antigens could not be detected . The ceramide part of the glycolipids was mainly composed of dihydroxy 18:0 long chain bases and non-hydroxy 16:0-24:0 fatty cids.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2319 - 25
Estimating the rate of plasmid transfer: an end-point method; Simonsen L et al.; We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture . The formula for estimating the plasmid transfer rate, gamma, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture . Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis . As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate gamma is unaffected by cell density, donor:recipient ratio and mating time . Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates . To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.

Electrophoresis, 1990 Nov, 11(11), 942 - 7
Free-flow electrophoresis for the purification of proteins: II . Isoelectric focusing and field step electrophoresis; Kuhn R et al.; Two modes of continuous isoelectric focusing are described . The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated . The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E . coli protein extract . Furthermore the principle of field step electrophoresis is presented . The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed . The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis . This approach permits a throughput of about 20 mL sample solution per minute.

Plasmid, 1990 Nov, 24(3), 195 - 207
The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum; Slade MB et al.; The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported . This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats . A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication . Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally . We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences . Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats . A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.

Biomed Environ Mass Spectrom, 1990 Nov, 19(11), 705 - 12
Analysis of post-translational modifications of proteins by accurate mass measurement in fast atom bombardment mass spectrometry; Takao T et al.; A rotatable dual-target probe was used for accurate mass measurement in fast atom bombardment mass spectrometry to determine the structures of unknown amino acid residues or post-translationally modified structures in peptides or proteins . The results obtained in measurement of tryptic peptides (with molecular weights of up to 2000) of the A-subunit of vero-toxin I indicated that the mass values obtained are sufficiently accurate and reproducible to allow the generation of the possible elemental compositions for modifications in peptides . By this method, the structural modifications of the N-terminal of a recombinant human leukocyte interferon A and novel halogenated amino acids in sperm-activating peptides from the egg-jelly of sea urchins were determined.

Genetika, 1990 Nov, 26(11), 1926 - 31
{Duplication in the region of the phoA and lacZ genes on the Escherichia coli K-12 chromosome}; Kulakauskas ST et al.; Transduction of genetic markers phoA1::Tn5 and lacZ::Tn10 was used to show the high frequency of occurrence of duplication (5-8%) carrying the above mentioned genes without application of selection for the increase of their function . Genetic tests were used to show that duplicated segments vary in size and in some cases comprise the whole lacZ-phoA region of Escherichia coli K-12 chromosome.

Microb Pathog, 1990 Nov, 9(5), 331 - 43
Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesion determinant: nucleotide sequence of the genes sfa B, C, D, E, F; Schmoll T et al.; The S fimbrial adhesin (sfa) determinant of E . coli comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA . Here the nucleotide sequence of the genes sfa B and sfa C situated proximal to the main structural gene sfaA is described . Sfa-LacZ fusions show that the two genes are transcribed in opposite directions . The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation studies indicated that the genes sfa B and sfa C play a role in regulation of the sfa determinant . In addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfa A and sfa G responsible for S subunit proteins, were determined . It is suggested that these genes are involved in transport and assembly of fimbrial subunits . Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1C fimbriae (foc) and type I fimbriae (fim) . The evolutionary relationship of fimbrial adhesion determinants is discussed.

FEMS Microbiol Lett, 1990 Nov, 60(3), 349 - 53
Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides; Watson GM et al.; A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides . Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000 . Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin . The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.

FEMS Microbiol Lett, 1990 Nov, 60(3), 329 - 35
The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE by ribosomal RNA gene restriction fragment patterns; Hookey JV; Deoxyribonucleic acid from Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE reference strains together with clinical and animal isolates were cleaved with EcoRI, electrophoresed and transferred to nylon membranes . An Escherichia coli MRE 600, 16S + 23S ribosomal RNA template was used in the synthesis of a single strand biotin-labelled cDNA probe using a reverse transcriptase . Ribosomal RNA cistrons present in the DNA fragments of strains were located using the cDNA probe . Between 4 and 7 well defined ribosomal RNA restriction bands were detected for the leptospires studied that varied in size from 12.1 to 0.57 kb . The hybridisation patterns were distinctive and unique for the reference strains that allowed for their characterisation . Similar patterns were observed for I . budapest M 20, I . "icterohaemorrhagiae" Ictero I and the type strain, I . icterohaemorrhagiae RGA . Clinical isolates and a isolate from a rat were compared with reference strain DNA patterns and identified as being similar to I . copenhageni M 20, I . "icterohaemorrhagiae" Ictero I and I . icterohaemorrhagiae RGA . These results indicate that ribosomal DNA fingerprinting may be useful in the classification and molecular epidemiology of pathogenic leptospires.

FEMS Microbiol Lett, 1990 Nov, 60(3), 289 - 92
Identification of asialo GM1 as a binding structure for Escherichia coli colonization factor antigens; Oro HS et al.; We have examined the binding of colonization factor antigens, (CFAs) of enterotoxigenic Escherichia coli to gangliosides and asialo gangliosides by using immuno-thin layer chromatography . CFA/II and its subcomponents (CS1, CS2 and CS3) as well as the subcomponent CS4 of the CFA/IV complex bound to asialo ganglioside GM1.

Biull Eksp Biol Med, 1990 Nov, 110(11), 529 - 31
{Pili determined by the tra genes of F-like plasmids}; Shchipkov VP et al.; The study of structural and functional features of plasmid-specific pili synthetized by E . coli cells under control of 27 F-like plasmids was performed . All the plasmids determined the pili of "flexible" type which were classified into 3 groups on the basis of difference in cell sensitivity to pili-specific phages f1, f2, Q . The possible role of transposons in the variation of pili functional properties was supposed.

Vet Microbiol, 1990 Nov, 25(2-3), 267 - 81
Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs; Bertschinger HU et al.; Two strains of E . coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment . Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E . coli (ETEC) . Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC . It adhered in vitro to porcine enterocyte brush border fragments . Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments . However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

Circ Shock, 1990 Nov, 32(3), 173 - 88
Effect of dietary alpha-linolenic acid on equine monocyte procoagulant activity and eicosanoid synthesis; Henry MM et al.; To investigate the effects of an omega-3 fatty acid-enriched ration on the in vitro response of equine monocytes to endotoxin, an 8-week feeding trial was conducted in which linseed oil served as the source of the omega-3 fatty acid, alpha-linolenic acid . One group of horses was fed a control pelleted ration and the other group was fed an 8% linseed oil-enriched pelleted ration . After 8 weeks of feeding, monocytes were isolated and incubated in the presence of Escherichia coli O55:B5 endotoxin for 6 hr . After 8 weeks on the rations, the mean procoagulant activity and thromboxane B2 production by endotoxin-stimulated monocytes from horses consuming the linseed oil ration decreased by 51% and 71%, respectively, compared with cells from horses consuming the control ration . There was no difference in monocyte synthesis of 12-hydroxyeicosatetraenoic acid or leukotriene B4 between groups . Fatty acid analysis of membrane phospholipids revealed a decrease in the omega-6:omega-3 ratio in monocytes from horses consuming the linseed oil ration . These data suggest that dietary supplementation with alpha-linolenic acid may modify the response to endotoxin by reducing the synthesis of potentially harmful cellular mediators.

Genetics, 1990 Nov, 126(3), 505 - 17
Molecular evolution of the Escherichia coli chromosome . III . Clonal frames; Milkman R et al.; PCR fragments, 1500-bp, from 15 previously sequenced regions in the Escherichia coli chromosome have been compared by restriction analysis in a large set of wild (ECOR) strains . Prior published observations of segmental clonality are confirmed: each of several sequence types is shared by a number of strains . The rate of recombinational replacement and the average size of the replacements are estimated in a set of closely related strains in which a clonal frame is dotted with occasional stretches of DNA belonging to other clones . A clonal hierarchy is described . Some new comparative sequencing data are presented.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8462 - 6
Evidence for positive and negative regulation of the Hox-3.1 gene; Bieberich CJ et al.; The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan . A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined . The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos . At 10.5 days p.c . and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia . Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia . Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element.

Metabolism, 1990 Nov, 39(11), 1180 - 5
Role of insulin in the blunted glucose metabolic response of septic rats to epinephrine; Hargrove DM et al.; Epinephrine produces smaller incremental increases in plasma glucose concentration and rate of glucose appearance (Ra) in septic rats compared with nonseptic animals . In the present study, we investigated the role of insulin in the diminished response of septic rats to epinephrine-induced increases in glucose turnover . Glucose kinetics were assessed by the infusion of {6-3H}-glucose in conscious catheterized rats made septic by subcutaneous injections of live Escherichia coli . Epinephrine was infused at 1 micrograms/min/kg for 2 hours in the presence and absence of somatostatin and mannoheptulose (SRIF + MH) . In comparison to nonseptic control animals, epinephrine-induced increases in plasma glucose concentration and glucose Ra were blunted by more than 50% in the septic rats . Infusion of SRIF + MH with epinephrine restored the blunted response to normal . During the infusion of epinephrine alone, the plasma insulin concentration in the septic rats was 2.8-fold higher than the nonseptic controls . SRIF + MH lowered the plasma insulin concentrations in both the nonseptic and septic rats to less than 10 microU/mL . SRIF + MH reversed the sepsis-induced hyperglucagonemia, but did not prevent a slight increase in glucagon levels during the epinephrine infusion in the nonseptic rats . In a second study, septic rats infused with SRIF + MH and replacement insulin showed a smaller increase in glucose concentration and glucose production in response to epinephrine than did septic animals administered SRIF + MH and no insulin . These results indicate that insulin plays an important role in the diminished response of septic rats to epinephrine.

J Bacteriol, 1990 Nov, 172(11), 6581 - 4
Cloning of an aroF allele encoding a tyrosine-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; Weaver LM et al.; In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively . A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.

Infect Immun, 1990 Nov, 58(11), 3594 - 600
Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli; Perez-Casal J et al.; Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium . We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one) . Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns . Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis . At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen . However, CS1-specific antisera did not react with CfaB . No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.

Protein Expr Purif, 1990 Nov, 1(2), 169 - 76
Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent; Lin K et al.; A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction . In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase . We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase . Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form . When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced . The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures . On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C . Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E . coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1990 Nov-Dec, 24(6), 1549 - 61
{Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates}; Danilevich VN et al.; In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained . The latter are equal or multiple of 9 b.p . in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon . The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame . One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates . For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1 . It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E . coli . However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon . Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained . It was shown that in the E . coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e . they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X . The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.

Mol Biol Rep, 1990 Nov, 14(4), 223 - 9
Detection of free cytoplasmic circles of transposon Tn9 multimers in Escherichia coli; Sarkari JF et al.; Extrachromosomal circular DNA molecules consisting of IS1-cat repeats, (IS1-cat)n, were isolated from an E . coli strain harboring nearly 30 copies of tandemly amplified transposon Tn9 located on the chromosome . The DNA 'circles' were characterized by restriction analysis followed by Southern blotting and electron microscopic examination . Their size varied from approximately 5.5 kb to 53 kb.

Res Immunol, 1990 Nov-Dec, 141(9), 879 - 92
Conformational change in the N-terminal domain of the Escherichia coli tryptophan synthase beta 2 subunit induced by its interactions with monoclonal antibodies; Blond-Elguindi S et al.; A fluorescence energy transfer signal was used to follow conformational changes occurring in two types of protein-protein complexes . The first complex studied was the native-like beta 2 subunit of Escherichia coli tryptophan synthase reconstituted by reassembly of the N- and C-terminal proteolytic domains of the beta chain . The other complexes were formed by the association of the N-terminal fragment (F1) with a monoclonal antibody that recognizes the native dimeric protein; four such complexes, obtained with different antibodies that recognize four distinct antigenic sites on native beta 2, were investigated . It was shown that a structural readjustment, which the isolated F1 domain was unable to undergo alone, was imposed upon F1 by interdomain interactions . Furthermore, with three of the four antibodies studied, the same conformational change in F1 also occurred after formation of the F1-antibody complex . These results demonstrate that, through an "induced fit mechanism", antigen-antibody stereospecific assembly can force the polypeptide chain to adopt a structure more closely resembling the conformation it has in the native protein.

Mol Biol Rep, 1990 Nov, 14(4), 247 - 9
Inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate allows estimation of the primers affinity; Podust VN et al.; Template-primer dependent inactivation of human DNA polymerase alpha and Klenow fragment of E . coli DNA polymerase I by adenosine 2',3'-riboepoxide 5'-triphosphate was used for quantitative analysis of the Kd values for oligonucleotide primers of different length . The Kd values are smaller by a factor of 2.5 than the Km values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase alpha . The Kd and Km values are nearly the same for Klenow fragment . Such approach to the determination of Km/Kd ratio can likely be used for detailed quantitative analysis of DNA polymerases.

Biochimie, 1990 Nov, 72(11), 813 - 8
RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA; Ost KA et al.; Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors . It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products . Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules . The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme . The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi . Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function . RNA chains as long as 40 residues can be synthesized in this system . As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.

Biochimie, 1990 Nov, 72(11), 791 - 802
Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+; Srivastava RK et al.; A precursor to 10Sa RNA accumulates in an rne mutant . However, the present studies indicate that RNase III is the enzyme that processes this RNA . Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III . That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain . It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions . In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane . Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment . The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA . The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation . The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation.

Mol Microbiol, 1990 Nov, 4(11), 1807 - 18
Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells; Gerdes K et al.; The hok/sok system of plasmid R1, which mediates plasmid stabilization via killing of plasmid-free segregants, encodes two genes: hok and sok . The hok gene product is a potent cell-killing protein . The expression of hok is regulated post-transcriptionally by the sok gene-encoded repressor, an antisense RNA complementary to the hok mRNA leader region . We show here that the hok mRNA is very stable, while the sok RNA decays rapidly . We also observe a new hok mRNA species which is 70 nucleotides shorter in the 3'-end than the full-length hok transcript . The appearance of the truncated hok mRNA was found to be regulated by the sok antisense RNA . Furthermore, the presence of the truncated hok mRNA was found to be correlated with efficient expression of the Hok protein . On the basis of these findings, we propose an extended model in order to explain the killing of plasmid-free segregants by the hok/sok system.

AIDS Res Hum Retroviruses, 1990 Nov, 6(11), 1297 - 303
Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT); Unge T et al.; The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted . The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form . Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique . The present crystal quality is still not adequate for high resolution X-ray investigation.

Acta Otolaryngol, 1990 Nov-Dec, 110(5-6), 410 - 5
Cholesteatoma debris as an activator of human monocytes . Potentiation of the production of tumor necrosis factor; Iino Y et al.; Tumor necrosis factor (TNF) is a cytokine which stimulates osteoclastic bone resorption and inhibits collagen synthesis in vitro . In this study the effect of human cholesteatoma debris and its constituents on the production of TNF-alpha by human monocytes in vitro was studied . Cultured human peripheral monocytes secreted TNF into the culture medium when exposed to cholesteatoma debris in a dose-dependent manner . The TNF production, however, was partially inhibited by the treatment of the debris with polymyxin B which inhibits biological activities of lipopolysaccharide (LPS) . When individual constituents of cholesteatoma debris, i.e . keratin, cholesterol, lauric acid and LPS, were added to the cultured monocytes at concentrations equivalent to those in the debris, significant production of TNF was observed only with the keratin and LPS . These data suggest that cholesteatoma debris is a potent activator of the TNF production of human monocytes in vitro, and that LPS and keratin are responsible for the production.

Biochem Cell Biol, 1990 Nov, 68(11), 1262 - 7
Antigenic determinant of a monoclonal antibody: extracellular domain at the M3-M4 junction of the alpha-subunit of Na,K-ATPase; Kano I et al.; The binding site of a monoclonal antibody, M45-80, against the alpha-subunit of horse Na,K-ATPase was determined . Various sizes of DNA fragments derived from rat Na,K-ATPase alpha 1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18 . Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens . An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse alpha 1-subunits) at the M3-M4 junction located on the extracellular side . The ouabain-binding site is discussed in relation to the recognition site of M45-80.

Scand J Immunol, 1990 Nov, 32(5), 537 - 44
The occurrence and sources of natural antibody in human bile and serum against the O antigens of two Escherichia coli serotypes; Hansen PG et al.; Paired serum and bile samples from normal subjects as well as patients with biliary disease were tested for natural antibody to two individual Escherichia coli O antigens by ELISA . Serum antibody was most commonly of IgM and IgG class . Antibody was less frequently detected in bile and was more commonly IgM than IgA, with IgG activity detected infrequently . Little relation was apparent between antibody in paired samples; activity could be present in both serum and bile or in either fluid alone . Titres in paired samples also did not correspond when 'normalized' with respect to the concentrations of relevant isotypes; bile was frequently enriched for natural antibody as a proportion of total immunoglobulin compared with serum . Secretory component-bound antibody was detectable in some biles that contained IgA and/or IgM activity and in the serum of 33% of subjects with biliary disorders but not in normal sera . A series of paired samples taken from three individuals was also examined for antibody against each subject's own intestinal commensal E . coli . Serum IgM and IgG activity was present in all samples, but antibody in bile was less frequent and was of IgM or IgA class . These results suggest that natural antibody in human bile occurs independently of antibody in serum and that it is substantially derived from local sources.

J Gen Virol, 1990 Nov, 71 ( Pt 11), 2665 - 72
The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens; Rayment FB et al.; We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli . These include native VP1 (84K) and VP2 (58K) proteins and also fusions to beta-galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide . Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble . This soluble polypeptide, p132, is a beta-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1 . Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples . We conclude that this region contains epitopes which must be prominently exposed on the intact virus . We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples . In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay . The recombinant p132 antigen is efficiently produced and readily purified from E . coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

J Cell Biol, 1990 Nov, 111(5 Pt 1), 2129 - 38
Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue; Burgess WH et al.; The fibroblast or heparin-binding growth factors (HBGFs) are thought to be modulators of cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction . A better understanding of the structural basis for the different activities of these proteins should facilitate the development of agonists and antagonists of specific HBGF activities and identification of the signal transduction pathways involved in the mechanisms of action of these growth factors . Chemical modification studies of Harper and Lobb (Harper, J . W., and R . R . Lobb . 1988 . Biochemistry . 27:671-678) implicated lysine 132 in HBGF-1 (acidic fibroblast growth factor) as being important to the heparin-binding, receptor-binding, and mitogenic activities of the protein . We changed lysine 132 to a glutamic acid residue by site-directed mutagenesis of the human cDNA and expressed the mutant protein in Escherichia coli to obtain sufficient quantities for functional studies . Replacement of this lysine with glutamic acid reduces the apparent affinity of HBGF-1 for immobilized heparin (elutes at 0.45 M NaCl vs . 1.1 M NaCl for wild-type) . Mitogenic assays established two points: (a) human recombinant HBGF-1 is highly dependent on the presence of heparin for optimal mitogenic activity, and (b) the change of lysine 132 to glutamic acid drastically reduces the specific mitogenic activity of HBGF-1 . The poor mitogenic activity of the mutant protein does not appear to be due to a reduced affinity for the HBGF receptor . Similarly, the mutant HBGF-1 can stimulate tyrosine kinase activity and induce protooncogene expression . Differences in the biological properties of the wild-type and mutant proteins were observed in transfection studies . Mutant HBGF-1 expression in transfected NIH 3T3 cells did not induce the same transformed phenotype characteristic of cells expressing wild-type HBGF-1 . Together these data indicate that different functional properties of HBGF-1 may be dissociated at the structural level.

J Cell Biol, 1990 Nov, 111(5 Pt 1), 1793 - 802
The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain; Romisch K et al.; Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V . R . Lingappa . 1986 . Annu . Rev . Cell Biol . 2:499-516) . It consists of SRP7S RNA and six proteins . The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides . The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle . We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle . Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA . Further deletions abolish SRP19 binding to SRP7S RNA . The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro . Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay . The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA . We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

J Bacteriol, 1990 Nov, 172(11), 6551 - 6
Physical analysis of phr gene transcription in Escherichia coli K-12; Lorence MC et al.; The phr gene of Escherichia coli K-12 encodes the light-dependent, DNA repair enzyme photolyase, which removes UV light-induced pyrimidine dimers from cellular DNA . From Southern hybridization analysis of several strains containing successively extended phr deletions, we have determined the direction of transcription of the phr gene on the E . coli K-12 chromosome . Northern (RNA) hybridization analysis suggests that the phr gene is cotranscribed with a previously identified gene of unknown function (orf169) into two messages of different lengths . S1 nuclease mapping analysis indicates that the two transcripts share a single termination site but initiate at two different sites . Finally, we have determined that the presence of orf169 is not necessary for phr gene activity in vivo.

J Bacteriol, 1990 Nov, 172(11), 6459 - 68
Primary sequence of the Escherichia coli fadBA operon, encoding the fatty acid-oxidizing multienzyme complex, indicates a high degree of homology to eucaryotic enzymes; DiRusso CC; In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in alpha 2 beta 2 conformation (A . Pramanik et al., J . Bacteriol . 137:469-473, 1979) . In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined . The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S . K . Spratt et al., J . Bacteriol . 158:535-542, 1984) . Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon . The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length . The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively . Recently, the sequence of the fadA gene was published in a separate report (Yang et al., J . Biol . Chem . 265:10424-10429, 1990) . In this work, most of the DNA sequence for fadA was confirmed, and 10 errors were corrected . Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide . By comparison to other peptide sequences, the alpha subunit encoded within fadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides . In agreement with the work of Yang et al., the beta subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide.

J Bacteriol, 1990 Nov, 172(11), 6175 - 81
Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli; Herzer PJ et al.; Multicopy single-stranded DNA (msDNA), a branched DNA-RNA molecule, has been shown in Escherichia coli B and clinical strain Cl-1 to be synthesized by reverse transcriptase . We report that 13% of the strains of the ECOR collection, a sample of 72 E . coli isolates representing the breadth of genetic variation of the species, produce msDNA . Three of the four major subspecific groups include msDNA-producing strains . Screening of 25 isolates that are genetically related to msDNA-producing clinical strains uncovered 22 additional msDNA-producing strains . A phylogenetic tree based on allelic variation detected electrophoretically at 20 enzyme-encoding loci revealed two major clusters and several deep branches composed of strains that synthesize msDNA . Although E . coli K-12 does not harbor msDNA, other closely related strains of the K-12 family do . The results support the hypothesis that msDNA-synthesizing systems, including reverse transcriptase genes, were acquired recently and independently in different lineages of E . coli.

Crit Care Med, 1990 Nov, 18(11), 1261 - 8
Ringer's acetate and dextran-70 with or without hypertonic saline in endotoxin-induced shock in pigs; Kristensen J et al.; The effects of Ringer's acetate, 6% dextran-70, 7.5% NaCl, and the combination of 7.5% NaCl and dextran-70 were tested in resuscitation from endotoxin shock induced by continuous iv infusion of Escherichia coli endotoxin in pigs . After about 3 h, a reproducible shock state was achieved and treatment was started, governed by the left atrial pressure . The hypertonic solutions (7.5% NaCl and 7.5% NaCl in dextran-70) did not show any overall advantages over the isotonic solutions (Ringer's acetate and dextran-70) . Only transient beneficial hemodynamic effects lasting less than 30 min after infusion were seen . When dextran-70 was administered, cardiovascular function was markedly improved and oxygen delivery (DO2) and survival were significantly higher compared with the crystalloid groups (Ringer's acetate and 7.5% NaCl) . Administration of large amounts of Ringer's acetate resulted in an immediate deterioration of pulmonary function . It was difficult to elevate left atrial pressure or even to keep it at baseline level, and cardiac index was only transiently increased . The overall result was a deterioration of DO2 and poor survival compared with the dextran-70 treated pigs . We conclude that dextran-70 is superior to Ringer's acetate in resuscitation from endotoxin-induced shock in pigs . Furthermore, we found no role for the use of hypertonic saline, alone or in combination with dextran, in the treatment of this type of prolonged endotoxin shock.

Neuron, 1990 Nov, 5(5), 569 - 81
A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties; Nakanishi N et al.; We have isolated two cDNA clones (GluR-K2 and GluR-K3) that share considerable sequence identity with the previously described glutamate receptor subunit, GluR-K1 . The three glutamate receptor subunits show significant sequence conservation with the glutamine binding component of the glutamine permease of E . coli . Each of these clones encodes a channel responsive to both kainate and AMPA . The coexpression of GluR-K2 with either GluR-K3 or GluR-K1 results in the formation of channels whose current-voltage relationships differ from those of the individual subunits alone and more closely approximate the properties of kainate receptors in neurons . These observations indicate that the kainate/quisqualate receptors are encoded by a family of genes and are likely to be composed of hetero-oligomers of at least two distinct subunits.

EMBO J, 1990 Nov, 9(11), 3787 - 94
Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters; Aldea M et al.; The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate . These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence . Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size . This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

EMBO J, 1990 Nov, 9(11), 3777 - 85
Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing; Persson C et al.; The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA . Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT . We have previously shown that the in vitro formation of the CopA-CopT duplex follows second-order kinetics and occurs in at least two steps . The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex . Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex . Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k-1) for the kissing complex (which are inferred to be the same for CopI-T and CopA-T), and measured the hybridization rate constant k2 (for CopA-T k2 is at least 1000-fold greater than for CopI-T) . The analysis of CopA derivatives of mutant and wild-type origin shows that the rate of formation of the kissing complex is rate-limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo . The biological implications of the kinetically irreversible RNA-RNA binding reaction scheme are discussed.

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