Escherichia coli

Can J Microbiol, 1992 Jul, 38(7), 699 - 704
Escherichia coli serotyping and disease in man and animals; Orskov F et al.; Serotyping of Escherichia coli is useful, but complex, with 173 O antigens, 80 K antigens, and 56 H antigens, which can all be subdivided into partial antigens . The O, K, and H antigens can be found in nature in many of the possible combinations . The final number of E . coli serotypes is very high, 50,000-100,000 or more . The number of frequent pathogenic serotypes is, however, limited . Two main groups of such frequent serotypes are (i) serotypes from diarrhoeal disease and (ii) serotypes from extraintestinal disease . Serotypes from diarrhoeal diseases are mostly species specific, and could at present be used as epidemiological markers for bacterial clones equipped with special virulence markers, such as toxins and adhesins . Their O-antigen lipopolysaccharides may be regarded as virulence factors . These strains are not inhabitants of the normal intestine . Serotypes from extraintestinal diseases constitute a different set of clones, which are good colonizers of the intestinal tract, that under certain conditions succeed in invading host tissues . They are characterized by virulence factors different from those found in strains from diarrhoeal disease . Thus, the two groups of pathogenic E . coli are both composed of a limited number of clones for which the O:K:H serotypes are excellent, although not faultless, markers.

Agents Actions, 1992 Jul, 36(3-4), 312 - 6
Lobenzarit disodium (CCA) inhibits the proliferation of human endothelial cells and the activity of DNA polymerase alpha; Takeuchi F et al.; N-(2)-Carboxyphenyl-4-chloroanthranilic acid disodium {Lobenzarit disodium (CCA)} is widely used for the treatment of patients with RA in Japan; however, the pharmacological mechanism of the compound is still unclear . In this report, the effect of CCA on the proliferation and DNA synthesis of endothelial cells was examined . CCA inhibited DNA synthesis in endothelial cells at a rather lower concentration than that in fibroblasts and HeLa cells . The DNA polymerase alpha activity was inhibited by CCA at a lower concentration than E . coli DNA polymerase I and avian myeloblastosis virus reverse transcriptase . Thus, CCA is a potent inhibitor of DNA polymerase alpha and this inhibitory effect could cause the inhibition of endothelial cell proliferation, which may be related to the therapeutic and pharmacological mechanisms of CCA.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 31 - 6
Synthesis of unusual thick pili by Escherichia coli of EPEC serogroup O119; Bradley DE et al.; Unique very thick pili were found in varying numbers on cells of five out of 11 clinical isolates of enteropathogenic Escherichia coli belonging to the classic EPEC serogroup O119 . They were approximately 12.5 nm in diameter, which is thicker than any other known E . coli pilus type . Analysis of the plasmid profiles of the O119 isolates showed that one strain was plasmid-free while the remainder contained numerous plasmids with a wide range of sizes . The thick pilus determinants were located on a 140-kb non-transferrable plasmid . They were not associated with adherence or a putative 90-kb enteroadherence factor plasmid.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 149 - 53
Bovine Escherichia coli of serotypes O55:H4 and O55:H21 which produce CNF2 express P fimbriae and Vir surface antigen, respectively; Blanco J et al.; We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55 . Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans . In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen . One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination.

Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 547 - 54
Flow cytometric analysis of protease activities in vital cells; Rothe G et al.; The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors . Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells . Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli . A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity . Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates . The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e . cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110 . Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64 . The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110 . Specificity was shown by inhibition with diisopropylfluorophosphate.

Anal Biochem, 1992 Jul, 204(1), 90 - 5
Application of 3-{3-(3-(trifluoromethyl)diazirin-3-yl)phenyl}-2,3- dihydroxypropionic acid, carbene-generating, cleavable cross-linking reagent for photoaffinity labeling; Bochkariov DE et al.; N-Hydroxysuccinimide ester of 3-{3-(3-(trifluoromethyl)diazirin-3-yl)phenyl}-2,3-dihydroxypro pionic acid was successfully tested in a ribosomal tRNA binding system . It is an originally designed trifluoromethyl-diazirine-based cleavable cross-linking reagent with a very short distance between the active points (about 8.5 A) . The reagent was coupled to the amino acid amino group of Phe-tRNAPhe to obtain a photoactivatable analog of peptidyl-tRNA . This analog was bound to ribosomes and the complex was irradiated with uv light . After isolation, the cross-linked product was cleft by periodate treatment to reveal the properties of the new reagent.

Antimicrob Agents Chemother, 1992 Jul, 36(7), 1441 - 6
Identification of the human immunodeficiency virus reverse transcriptase residues that contribute to the activity of diverse nonnucleoside inhibitors; Condra JH et al.; The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds . These include pyridinone derivatives, tetrahydroimadazo{4,5,1-j,k}{1,4}-benzodiazepin-2(1H)-one and -thione, and BI-RG-587 (nevirapine) . The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates . Despite a high degree of similarity between the HIV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors . Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities . The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined . Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188 . The region defined by residues 101 to 106 was found to functionally interact with the domain from 155 to 217 . In addition, the functional equivalence of the three inhibitor groups was shown.

Mol Gen Genet, 1992 Jul, 234(1), 121 - 8
Localised mutagenesis of the fts YEX operon: conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients; Gibbs TW et al.; After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants . Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX, whereas others were defective in the heat shock regulator gene rpoH . Both missense and amber mutant alleles of these genes were produced . The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site . Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein . This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man . One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients . These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5779 - 83
Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogen-activated protein kinase p42mapk; Rossomando AJ et al.; Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source . Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E . coli and during in vitro incubations with ATP/Mg2+/Mn2+ . Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells.

Mutat Res, 1992 Jul, 282(3), 203 - 7
Inhibition of recA induction by the radioprotector 2-mercaptoethylamine; Naslund M et al.; Our earlier finding that the radioprotective action of 2-mercaptoethylamine (MEA) is counteracted by ascorbate suggests a biochemical mechanism of action, which is supported by observations that MEA is not radioprotective in Rec- E . coli strains . In this study we show that MEA inhibits the induction of the recA gene by UV- or gamma-irradiation or by nalidixic acid in Escherichia coli strain GE94, which contains a recA-lacZ fusion . This effect, which may be counteracted by cysteine, indicates that in general MEA inhibits the induction of SOS functions.

Mutat Res, 1992 Jul, 282(3), 183 - 90
Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli; Okaichi K et al.; A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B . Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene . Plasmid survival was also measured by transforming activity of the indicator strain . The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%) . Some hot-spots in the mutations were observed by sequencing the suppressor gene . Mutagenic specificity in DNA base sequences induced by HL in E . coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells . This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.

Eur J Biochem, 1992 Jul 1, 207(1), 305 - 13
Molecular cloning, stage-specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum; Zhao Y et al.; A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases . The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A+T)-rich region and transcribed in a different orientation . No intron sequence exists in PfPK2 . The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases . Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine cAMP-dependent protein kinase, respectively . Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits . Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane.

EMBO J, 1992 Jul, 11(7), 2675 - 83
Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation; Blomberg P et al.; The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA . We present results demonstrating that CopA controls RepA synthesis indirectly . A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes . A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA . Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression . Specific suppression of the stop codons reversed the effect . This indicates that tap translation is required for RepA synthesis . Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation . The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation . The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1992 Jul, 11(7), 2627 - 32
The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli; Jordi BJ et al.; CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C . Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid . Region 2 encodes a protein (CfaD) which activates the promoter in region 1 . We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae . As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression . CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C . More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain . This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein . We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.

EMBO J, 1992 Jul, 11(7), 2457 - 63
The aerolysin membrane channel is formed by heptamerization of the monomer; Wilmsen HU et al.; The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals . Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids . A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter . The ring is surrounded by seven arms each made up of two unequal sized domains . By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed . The relevance of this structure to the mode of action of aerolysin is discussed.

Mutat Res, 1992 Jul, 268(1), 59 - 64
Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions; Matsumoto K et al.; N4-Aminocytidine is mutagenic in various organisms . In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA . To prove that the N4-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA . We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations . The M13mp2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies . Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present.

J Bacteriol, 1992 Jul, 174(14), 4558 - 75
TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization; Wilmes-Riesenberg MR et al.; We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene . Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene . We describe three kinds of elements for making each fusion type . Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance . The third kind is transposition defective (Tnp-) and encodes kanamycin resistance . In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions . Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances . Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype . Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments . Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription . This feature is especially useful in the determination of operon structures . Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described.

Biotechnology (N Y), 1992 Jul, 10(7), 795 - 8
Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli; Ortega S et al.; We describe a method for the purification of recombinant proteins based upon the selective interaction of the choline-binding domain of the pneumococcal murein hydrolase and tertiary amines . Proteins of interest, fused to the binding domain by a peptide linker, containing the cleaving sequence recognized by blood coagulation factor Xa, can either be assayed for biological activities in vitro and in vivo or have the binding moiety removed to yield a totally unmodified form, suitable for clinical and functional studies . The method can also be applied to the production of low molecular mass peptides . The principle of the technique is illustrated with acidic fibroblast growth factor and with a neuropeptide-like fragment of ten amino acids contained within its sequence.

Blood, 1992 Jul 1, 80(1), 170 - 8
Infection of hematopoietic progenitor cells by human cytomegalovirus; Maciejewski JP et al.; The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256 . RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter . Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells . Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies . By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies . LacZ activity was observed predominantly in cells of myelomonocytic lineage . When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered . Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation . Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.

J Immunol, 1992 Jul 1, 149(1), 113 - 9
Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo . Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells; Colombo MP et al.; We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection . The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay . We present direct evidence that neutrophils are involved in this phenomenon . We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c . injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice . The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells . We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells . To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios . Histologic and EM analysis of the tumors growing at the site of injection were carried out . Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells . In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils . In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells . Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition . The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present.

J Biotechnol, 1992 Jul, 24(3), 235 - 51
Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation; Maschke HE et al.; Plasmid instabilities in E . coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions . In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions . The selective pressure against pRK248cI stabilized the pMTC48 . This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48) . In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions . This plasmid was stabilized in the host growing under selective conditions . During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells . Plasmid analysis from the above cells showed that the plasmid has undergone structural instability . Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities . Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.

Biosci Biotechnol Biochem, 1992 Jul, 56(7), 1012 - 6
Syntheses of recombinant yellowtail and flounder growth hormones in Escherichia coli; Watahiki M et al.; For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E . coli, expression plasmids were constructed . The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively . These product proteins were accumulated in inclusion bodies in the cells . The recombinant hormones were isolated from the pellets ina glutathione reduction/oxidation buffer . The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity . The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine . The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.

Biotechnology (N Y), 1992 Jul, 10(7), 790 - 4
Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco; Owen M et al.; We have expressed a synthetic gene that encodes an antigen-binding single-chain FV protein (scFV) in transgenic tobacco plants . The scFV gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line . The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome . The cloned scFV gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome-binding binding scFV protein . Transgenic tobacco plants expressing the scFV gene were also found to produce a functional scFV protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome-dependent germination . The scFV from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome-Sepharose affinity chromatography step.

Res Immunol, 1992 Jul-Aug, 143(6), 601 - 10
TNF alpha-mediated tissue damage in mouse footpads primed with mycobacterial preparations; al Attiyah R et al.; Tissue sites involved in certain types of inflammation become sensitive to the destructive effect of a subsequent injection of tumour necrosis factor alpha (TNF alpha) . To try to further delineate the cascade of effector and regulatory events controlling this activity, a new model is described and its main properties characterized . C57BL/GrFa mice received mycobacterial products subcutaneously in the footpads . Recombinant TNF alpha was injected 24 h later into the same sites . To assess the tissue-destructive effect of TNF alpha in these "primed" footpads, swelling and haemoglobin content of injected footpads were measured, 16 h and 20 h respectively after the injection of TNF alpha . When loaded with either Escherichia coli LPS (10 micrograms) or Mycobacterium vaccae soluble sonicate (17 micrograms), footpads were reactive to the subsequent injection of 1 microgram recombinant TNF alpha, as assessed by both swelling and haemoglobin content . When C57BL/GrFa mice received 10(9) autoclaved M . vaccae subcutaneously in the back 10 days before the footpad was "primed" with soluble M . vaccae sonicate, the destructive effect of TNF alpha was significantly enhanced, becoming 5-10-fold greater than that seen in sites "primed" with an optimal dose of LPS . This higher reactivity was abrogated by a single dose of anti-CD4 given just before the injection of TNF alpha . This local reactivity to TNF alpha of skin sites loaded with mycobacterial products is compared to the local LPS-dependent Shwartzman reaction, and the relevance of this assay as a model with which to delineate the mechanisms of tissue damage in tuberculosis is discussed.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1495 - 502
Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli; Harel J et al.; The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165) . The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established . On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated . The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa . Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads . Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.

Mol Microbiol, 1992 Jul, 6(14), 1995 - 2007
Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES; Rinke de Wit TF et al.; In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene . The complete DNA sequence of this groESL operon was determined . The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins . Southern blotting analyses with M . leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis . This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene . Using five T-cell clones from two leprosy patients as probes, expression of the M . leprae GroES protein in Escherichia coli after heat shock was demonstrated . Four of these clones recognized the same M . leprae-specific GroES-derived peptide in a DR2-restricted fashion . No expression of the groEL gene from this operon was detected in E . coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.

Int J Radiat Biol, 1992 Jul, 62(1), 21 - 32
Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells; Bases R et al.; The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells . Rat cells were chosen for transfection because they have no alpha DNA . Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation . The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments . alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection . The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection . Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection . These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min . The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair . Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E . coli . Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al . 1990).

J Bacteriol, 1992 Jul, 174(14), 4538 - 48
Characterization of Escherichia coli glnL mutations affecting nitrogen regulation; Atkinson MR et al.; Nitrogen regulator II (NRII), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI . Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NRI-phosphate phosphatase activity that is observed when NRII and another signal transduction protein known as PII (the glnB product) interact . The availability of PII for this interaction is controlled by a uridylytransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies PII in response to intracellular signals of nitrogen availability . Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation . The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains . Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NRII autophosphorylation at His-139 . Complementation experiments with multicopy plasmids encoding NRII or PII showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NRII but was not affected by introduction of the plasmid encoding PII . Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NRII or that encoding PII . We hypothesize that mutants of the latter type result in a subtle perturbation of the NRII-PII interaction and suggest two possible mechanisms for their effects.

Infect Immun, 1992 Jul, 60(7), 2828 - 34
Passive protection of suckling infant mice against F41-positive enterotoxigenic Escherichia coli strains by intravenous inoculation of the dams with monoclonal antibodies against F41; Duchet-Suchaux M et al.; Ten monoclonal antibodies (MAbs) against five different epitope clusters of adhesion factor F41 (two MAbs per cluster) were tested for protection of infant mice against an oral challenge with F41-positive enterotoxigenic Escherichia coli (ETEC) B2C and B41M . Infant mice suckling dams intravenously inoculated with MAbs were orally challenged, and the survival rates were measured for 12 days after inoculation and challenge . Irrespective of their epitope specificity, all F41 MAbs given in a single dose of 4 mg per dam had a protective effect against both ETEC strains . In contrast, one K99 MAb of the same isotype and given in the same dose as the F41 MAbs did not protect infant mice at all . A reduction in the dose of F41 MAbs to 0.032 mg per dam resulted in a decrease in protection . Two different MAbs against the same epitope cluster were not necessarily equally protective . Combining MAbs two by two, whether the MAbs recognized the same epitope cluster or not, resulted in protective activity essentially similar to that obtained with each MAb separately, without any improvement . Therefore, one MAb against any epitope may be sufficient for protection . Enzyme-linked immunosorbent assay (ELISA) titers of MAbs in the serum of dams were similar, irrespective of the epitope specificity of the MAbs, and gradually decreased from day 1 to day 12 after inoculation . We found a good correlation between colostrum and milk ELISA titers of MAbs and serum ELISA titers of MAbs . Colostrum and milk MAb titers were 10-fold lower than corresponding serum MAb titers and stayed high until day 5 after inoculation . The most protective MAb had the highest ELISA titers in colostrum and milk for the first 5 days after inoculation . ETEC strain B2C colonized the intestines of infant mice suckling MAb-inoculated mothers until day 12 after challenge . Intestinal levels of the challenge strain were high on day 2 but never reached the very high numbers (10(9) to 10(10)) described previously in a diarrheic infant mouse model . MAbs did not eliminate the challenge ETEC strain from the intestines of infant mice.

Infect Immun, 1992 Jul, 60(7), 2648 - 56
pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys; Dozois CM et al.; Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production . Serogroups O78 and O1 were most common in septicemic chickens and turkeys . pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks . Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin . The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys . Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated with pil+ isolates from turkeys . Serum resistance was associated with isolates from septicemic turkeys and with lethality in isolates from chickens . The aerobactin system was associated with isolates from septicemic chickens and turkeys . Overall, results indicated that (i) genotypic examination may reveal virulence-associated traits which differ from the typically expected phenotype and/or are not readily expressed in vitro, and (ii) certain phenotypic and genotypic traits associated with E . coli causing extraintestinal disease in humans and animals are also associated with E . coli causing avian septicemia.

Plant J, 1992 Jul, 2(4), 549 - 57
Potato virus X as a vector for gene expression in plants; Chapman S et al.; The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs . In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene . In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA . The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants . These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector . The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue . Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants . These data point to a general utility of PVX as a vector for unregulated gene expression in plants.

Arch Biochem Biophys, 1992 Jul, 296(1), 122 - 8
Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B; Ghosh P et al.; The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E . coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties . The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme . The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site . One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC . The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain . The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands . The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.

Protein Sci, 1992 Jul, 1(7), 910 - 6
Cis proline mutants of ribonuclease A . I . Thermal stability; Schultz DA et al.; A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol . 185, 60-89) . The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A . The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification . Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made . In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing . Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol . The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.

Biull Eksp Biol Med, 1992 Jul, 114(8), 204 - 5
{The molecular cloning of a genetic region determining the surface exclusion system of the F-like plasmid pAP42}; Krivskaia KS et al.; Molecular cloning of genetic region of F-like plasmid pAP42, coding its surface exclusion system (system Six V) was performed . Restriction and genetic analysis of recombinant plasmids showed that six V locus is situated in Sal I-fragment f5 (4.2 MD) of this plasmid.

J Biochem (Tokyo), 1992 Jul, 112(1), 57 - 62
Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle; Iwakura M et al.; Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C . Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity . Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein . The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated . However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer . The fusion protein was highly purified by means of a methotrexate affinity chromatography.

J Biochem (Tokyo), 1992 Jul, 112(1), 1 - 6
Activation of the osmoregulated ompF and ompC genes by the OmpR protein in Escherichia coli: a study involving chimeric promoters; Takayanagi K et al.; The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity . The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites . In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid . It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene . In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472) . It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur . These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein.

Development, 1992 Jul, 115(3), 729 - 35
Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster; Lachaume P et al.; The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster . Its mobility is associated with the I-R hybrid dysgenesis . In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies . Homozygous transgenic Drosophila lines have been established and analysed . ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis . No somatic expression is found . Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly . This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al . (Mizrokhi, L.J., Georgevia, S.G . and Ilying, Y.V . (1988) . Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor . The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein . This expression is restricted to dysgenic and reactive females, and is similar in the two conditions . All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level . It also appears that our transgene is an efficient marker to follow I factor expression.

J Pediatr Gastroenterol Nutr, 1992 Jul, 15(1), 63 - 72
Field trial of an infant formula containing anti-rotavirus and anti-Escherichia coli milk antibodies from hyperimmunized cows; Brunser O et al.; Two groups of 124 and 108 children, respectively, living in urban Santiago, Chile in low socioeconomic conditions were prospectively followed for 6 months for their incidence of diarrhea . Each cohort was divided into two subgroups receiving either a commercial milk formula or the same formula containing 1% (wt/wt) bovine milk immunoglobulin concentrate from cows hyperimmunized with human rotaviruses and the major enteropathogenic Escherichia coli (EPEC) serogroups . Neither group differed with respect to incidence of diarrhea (98 episodes in 117 treated children versus 95 episodes in 115 control children), duration and clinical symptoms of diarrhea, and weight gain . Furthermore, neither group differed with respect to isolation of rotavirus (14 and 13 isolates in treatment and control groups, respectively) and isolation of enteropathogenic E . coli (14 and 15 isolates in treatment and control groups, respectively) . The treatment but not the control formula contained neutralizing antibody against all human rotavirus serotypes . Titers were comparable to human breast milk samples . All isolated EPEC serogroups were included in the vaccine used for the immunization of the cows . The treatment, but not the control formula, protected mice against a lethal challenge with an EPEC strain . In conclusion, feeding an antibody-supplemented formula had no positive effect on diarrheal diseases under the conditions of a fairly well-controlled small-scale field trial.

Biokhimiia, 1992 Jul, 57(7), 1057 - 64
{Insertional mutagenesis of protease 2A of the poliomyelitis virus and its substrate, simultaneously expressed in Escherichia coli cells}; Slobodskaia OR et al.; In order to clarify the structural features of protein substrates which determine their sensitivity towards poliovirus 2A protease, a high-efficiency bacterial expression system for cDNA of the poliovirus genome fragment has been developed . The expressed protein encompasses the C-end half of the VP1 capsid protein . 2A protease, and a large portion of the 2B protein . Virus-specific products were found in the insoluble fraction of bacterial cell lysates which were mainly represented by two proteins . These proteins appeared to be produced via the cleavage of the expressed protein by 2A protease at the Tyr-Gly site located between the VP1 protein and the 2A protease proper . The accumulation of two viral proteins can be prevented by a four amino acid insertion into the 2A gene locus in close vicinity of the putative catalytic His residue . The determinants of specificity toward the 2A action are located within the region flanking the cleavable Tyr-Gly dipeptide from its N-side.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 1 - 6
Carbon monoxide-binding properties of the cytochrome bo quinol oxidase complex in Escherichia coli are changed by copper deficiency in continuous culture; Ciccognani DT et al.; A strain of Escherichia coli having elevated levels of cytochrome bo and lacking the cytochrome bd quinol oxidase was grown in chemostat culture at low copper levels . Such cells had lowered levels of copper and of total cytochrome b . Cytochrome o concentration was unchanged when assayed by conventional CO difference spectroscopy, but apparently diminished by 80% in copper-deficient cells as determined by photodissociation of bound CO at 193 K . This is attributed to depletion of copper in the oxidase of copper-deficient cells, causing rapid recombination of photodissociated CO to haem O . CO recombination was also more sensitive to low intensities of actinic light in copper-depleted oxidase . The results illustrate a further similarity between the active sites of o- and aa3-type terminal oxidases.

Plasmid, 1992 Jul, 28(1), 80 - 5
Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7; Flores CC et al.; The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E . Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7 . Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed . Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins . The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7 . The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7.

Jpn J Cancer Res, 1992 Jul, 83(7), 705 - 13
Identification of antibodies against human papillomavirus type 16 E6 and E7 proteins in sera of patients with cervical neoplasias; Sasagawa T et al.; We have developed a sensitive and specific ELISA method using the s10-fusion proteins of human papillomavirus (HPV) 16 E6 and E7, expressed in E . coli . Sera from 97 women (30 patients with invasive cervical cancers, 26 patients with cervical intraepithelial neoplasia III (CIN III) and 38 healthy women) were tested for the presence of antibodies to E6 and E7 proteins . Eight (27%) of the 30 cervical cancer sera, five (19%) of the 26 CIN sera and none of the 38 normal sera were reactive with E6 proteins (cut-off point: absorbance (A) = 0.59, x +3SD) . Ten (33%) of the 30 cervical cancer sera, two (8%) of the 26 CIN III sera and none of the 38 normal sera were reactive with E7 proteins (cut-off point: A = 0.40, x +3SD) . The mean absorbance for anti-E7 antibody in positive cases was higher in cancer patients than in CIN III patients, while that for E6 did not differ between these two groups . Interestingly, six (50%) of 12 cancer sera which reacted with either E6 or E7 proteins were reactive for both proteins, whereas none of the sera from the CIN III patients reacted with both proteins . The high prevalence rates and high absorbance values for HPV 16 E6 and E7 antibodies in association with malignant transformation suggest that detection of these antibodies may be a useful diagnostic aid for cervical cancer-associated HPV 16.

Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 523 - 8
Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants; Luderer M et al.; The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mp18 . The fusion of an active 2A proteinase to the C-terminus of the alpha-fragment did not affect alpha-complementation, as the proteinase cleaved itself off the alpha-fragment . However, an inactive 2A proteinase remained fused to the alpha-fragment hindering alpha-complementation . Random mutations were then introduced into the 2A gene site by PCR amplification . Mutants defective in alpha-complementation (thus containing an inactive 2A proteinase) were obtained at an efficiency of 5%, mutants showing reduced 2A activity at an efficiency of 1% . Mutants showing reduced or no 2A activity were then subjected to PCR mutagenesis . Three mutants reactivating an inactive 2A proteinase were examined and the compensatory changes determined.

Mol Microbiol, 1992 Jul, 6(14), 1877 - 86
Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli; Hengge-Aronis R et al.; The putative stationary-phase sigma factor (sigma S) encoded by rpoS is essential for glycogen synthesis, but is not required for the transcription of glgC and glgA, which encode ADP-glucose-pyrophosphorylase and glycogen synthase, respectively . Using a mini-Mu random chromosomal library and a screen for glycogen overproduction, we identified a novel gene (glgS) involved in glycogen synthesis . glgS maps at 66.6 min (3247 kb) on the chromosome and constitutes a monocistronic operon . It encodes a hydrophilic and highly charged small protein, with a molecular weight of 7886, which is strongly expressed in minicells . Experiments with single-copy chromosomal glgS::lacZ gene fusions indicated that glgS expression is controlled by sigma S as well as by cAMP . Two transcriptional start sites were mapped in the upstream regulatory region of glgS . The glgSp1 transcript was absent in a cya mutant, whereas an rpoS mutant did not synthesize the glgSp2 transcript . Although glycogen synthesis is strongly stimulated by overproduction of GlgS and is inhibited by a glgS null mutation, glgS does not affect the expression of the glgCAP operon . Its potential role in the metabolic control of glycogen synthesis is discussed . Also, evidence is presented to show that the amount of glycogen accumulated in vivo in early stationary-phase cells is mainly determined by sigma S-controlled gene expression and allosteric activation of GlgC, whereas the absolute levels of expression of glgCAP as well as the intracellular concentration of cAMP are of minor importance.

J Virol Methods, 1992 Jul, 38(1), 81 - 92
Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus; Lee LH; Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome . The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichia coli strain JM109 . A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23 . The DNA fragment within pC23 was 320 base pairs in length and designated C23 . Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay . Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains . All four biotinylated probes detected three serotype 1 viruses and one serotype 2 IBDV . However, they did not cross-react with nucleic acids extracted from mock-infected cells or from seven unrelated avian viruses . Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA . In addition, the probe pC23/BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 91 - 7
Binding of the cyclic AMP receptor protein of Escherichia coli and DNA bending at the P4 promoter of pBR322; Brierley I et al.; The binding of the Escherichia coli cyclic AMP receptor protein (CRP) to its specific site on the P4 promoter of pBR322 has been studied by gel electrophoresis . Binding to the P4 site was about 40-50-fold weaker than to the principal CRP site on the lactose promoter at both low (0.01 M) and high (0.1 M) ionic strengths . CRP-induced bending at the P4 site was investigated from the mobilities of CRP bound to circularly permuted P4 fragments . The estimated bending angle, based on comparison with Zinkel & Crothers {(1990) Biopolymers 29, 29-38} A-tract bending standards, was found to be approximately 96 degrees, similar to that found for binding to the lac site . These observations suggest that there is not a simple relationship between strength of CRP binding and the extent of induced bending for different CRP sites . The apparent centre of bending in P4 is displaced about 6-8 bp away from the conserved TGTGA sequence and the P4 transcription start site.

Mol Microbiol, 1992 Jul, 6(13), 1829 - 39
PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA; Albano MA et al.; To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1) . Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen . Using MudphoA we generated fusions to an E . coli gene encoding a periplasmic protein and to an L . pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity . We have begun to use MudphoA to mutate secreted proteins of L . pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions . This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.

Mol Microbiol, 1992 Jul, 6(13), 1739 - 45
Differential regulation of two genes encoding lysyl-tRNA synthetases in Escherichia coli: lysU-constitutive mutations compensate for a lysS null mutation; Kawakami K et al.; Lysyl-tRNA synthetases are synthesized in Escherichia coli from two distinct genes, lysS and lysU, which are regulated differentially . A strain which is null for lysS, the constitutive gene, was created by gene disruption (lysS1) and exhibited cold-sensitive lethality . Hence, lysS is dispensable at high temperatures . This cold sensitivity was suppressed by a multi-copy plasmid carrying lysU, the inducible gene . These data are interpreted as indicating that lysS is functionally replaceable by lysU for cell growth, and that the cold sensitivity of lysS1 is caused by insufficient expression of lysU at low temperatures . To investigate the mechanism of lysU expression, cold-resistant bypass mutations were isolated from lysS1, and named als (for abandonment of lysS) . Two als mutations which were linked to lysU contain IS2 insertions upstream of the lysU promoter . They caused a 16-19-fold increase in the lysU-mRNA level . Furthermore, deletion mutations created immediately upstream of the lysU promoter restored growth of lysS1 . These results suggest that transcription of lysU is negatively controlled by a cis-element located upstream of the promoter.

J Cell Biol, 1992 Jul, 118(2), 227 - 44
Disulfide bond formation during the folding of influenza virus hemagglutinin; Segal MS et al.; To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues . Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues . We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

Eur J Biochem, 1992 Jul 1, 207(1), 61 - 8
EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli . Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism; Guigliarelli B et al.; The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy . A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials . The high-potential group comprises a {3Fe-4S} and a {4Fe-4S} cluster whose midpoint potentials are +60 mV and +80 mV, respectively . Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV . The {4Fe-4S}1+ center occurs in two different conformations as shown by its composite EPR spectrum . The low-potential group contains two {4Fe-4S} clusters with more typical redox potentials (-200 mV and -400 mV) . In the fully reduced state, the three {4Fe-4S}1+ centers are magnetically coupled, leading to a broad featureless spectrum . The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied . The iron-sulfur centers of the second nitrate reductase of E . coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV . From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.

Eur J Biochem, 1992 Jul 1, 207(1), 195 - 200
Enhanced cell-free transcription of the ribosomal protein L32 gene by the polyoma virus enhancer PEA3 DNA-binding protein; Yoganathan T et al.; The mouse-ribosomal-protein-L32-gene promoter contains a 12-bp sequence motif within the 5'-upstream region termed the beta element which shows significant similarity with the consensus sequence of the polyoma-virus-enhancer PEA3 . A cloned PEA3 DNA-binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal-protein-L32 gene in a cell-free system . Moreover, the PEA3 protein participates in the formation of the ribosomal-protein-L32-promoter-preinitiation-transcription complex . The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the beta element . In addition anti-PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the beta element, causing a supershift in a mobility-shift assay . Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell-free transcription system.

Eur J Biochem, 1992 Jul 1, 207(1), 177 - 83
A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor; Ballance DJ et al.; The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment . Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g . cancer and some inflammatory diseases . To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae . The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells . GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR . Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.

Am Rev Respir Dis, 1992 Jul, 146(1), 32 - 8
Dibutyryl cyclic AMP attenuates lung responses induced by endotoxin in conscious sheep; Koyama S et al.; Dibutyryl cyclic AMP (DBcAMP) could inhibit the production of prostanoids and modulate the pulmonary vascular responses induced by endotoxin . Diffuse lung injury after endotoxemia in sheep is accompanied by the production of prostanoids and an increase in endothelial permeability . To determine whether exogenous DBcAMP could prevent the endotoxin responses, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to an intravenous infusion of Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) in unanesthetized sheep in the presence and absence of DBcAMP (30 micrograms/kg/min) infused intravenously for 6 h beginning 1 h before endotoxin infusion or for 4.5 h after 30 min of treatment with endotoxin infusion . We also measured circulating leukocytes and lung lymph and plasma concentrations of thromboxane B2 (TXB2) and prostacyclin (6-keto-PGF1 alpha) metabolites by radioimmunoassay . DBcAMP infusion before endotoxin infusion decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the increased lung lymph flow and lymph protein clearance . DBcAMP after endotoxin only attenuated the increased lung lymph flow and lymph protein clearance . DBcAMP treatment both before and after endotoxin infusion blocked endotoxin-induced increases in lung lymph and plasma TXB2 and 6-keto-PGF1 alpha . DBcAMP did not affect the number of circulating leukocytes . Although DBcAMP alone did not affect the pulmonary and systemic hemodynamics and lung lymph balance, the potential that DBcAMP directly modulates the pulmonary vascular responses to endotoxin as a vasodilator could be expected . We conclude that DBcAMP infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing prostanoid release and modulating the pulmonary vascular responses.

J Bacteriol, 1992 Jul, 174(14), 4856 - 9
Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides; Weissborn AC et al.; We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides . Four phenotypic classes were identified, including two with a novel array of characteristics . The mutations mapped to two genetic loci . Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides . Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.

J Bacteriol, 1992 Jul, 174(14), 4530 - 7
Fis plays a role in Tn5 and IS50 transposition; Weinreich MD et al.; The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition . Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria . This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins . We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited . The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate . Using a mobility shift assay, we also identified another potential Fis site within IS50 . Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.

J Bacteriol, 1992 Jul, 174(13), 4509 - 12
Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B; Meyer CR et al.; A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe . The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.

J Bacteriol, 1992 Jul, 174(13), 4212 - 7
Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange; Ferrero RL et al.; Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement . A region of cloned H . pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon . Electrotransformation of H . pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H . pylori transformants . Competence for electrotransformation appeared to be restricted to certain wild-type H . pylori isolates; only 1 isolate (of 10 tested) was consistently transformed . Two of the kanamycin-resistant H . pylori transformants were further studied and shown to be urease negative . Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector . Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H . pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product . Generation of stable, genetically engineered urease mutants of H . pylori will be useful for addressing the role of urease in the pathogenesis of H . pylori infection.

Cancer Res, 1992 Jul 1, 52(13), 3760 - 7
Oncogene complementation in fetal brain transplants; Wiestler OD et al.; Using a neural transplantation model and retrovirus-mediated gene transfer, we have introduced the oncogenes v-Ha-ras and v-myc into the developing rat brain . Upon insertion of a construct encoding v-Ha-ras and the Escherichia coli beta-galactosidase marker gene, the retroviral vector was found to be expressed in neurons, astrocytes, and endothelial cells of the graft . After latency periods of several months, fascicular neoplasms with expression of S-100 protein were observed in 50% of the transplants . The foreign genes were shown to be highly expressed in the tumors and in intact donor cells, by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry, indicating that an activated Ha-ras oncogene has the potential to initiate neoplastic transformation of glial cells . Introduction of the v-myc oncogene into 15 grafts resulted in only a single primitive neuroectodermal tumor . However, simultaneous expression of the v-Ha-ras and v-myc genes yielded highly malignant, polyclonal neoplasms in all recipient animals, as early as 13 days after transplantation, from which cell lines could be easily derived . In addition, neoplastic transformation was also observed in vitro following introduction of ras and myc into embryonic forebrain cultures and into newborn cerebellar cultures . These data indicate a powerful complementary transforming effect of ras and myc on neural progenitors in vivo and in vitro . Coexpression of ras and myc may, therefore, provide a highly efficient tool for transforming neural precursor cells in distinct segments of the central nervous system at different stages of development.

Infect Immun, 1992 Jul, 60(7), 2870 - 3
Binding of class II Escherichia coli enterotoxins to mouse Y1 and intestinal cells; Donta ST et al.; The binding of class II Escherichia coli heat-labile enterotoxins (LT) to Y1 tissue-cultured cells and mouse intestinal cells was studied and compared with that of class I toxins, including cholera enterotoxin . All radioiodinated (125I) toxins retained their biological activities in both model systems, but only LTIIb could be shown to bind specifically to target cells . LTIIa could inhibit the binding of both class I and LTIIb toxins, a finding which correlates with its ability to bind to multiple gangliosides . LTIIb could not inhibit the binding of the other enterotoxins . The binding and activity of class II toxins could not be modulated by prior exposure of target cells to the B subunit of LTI.

Infect Immun, 1992 Jul, 60(7), 2572 - 80
Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture; Kerneis S et al.; To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used . Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation . We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells . The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA . No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells . However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity . This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present . ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed . Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture . This indicated that the expression of the ETEC CFA receptors was a growth-related event . Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.

Curr Genet, 1992 Jul, 22(1), 41 - 5
Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum; Marmeisse R et al.; The pAN7.1 plasmid containing the E . coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum . Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA . Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome . The selection system was used to introduce other genes of interest by co-transformation . Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H . cylindrosporum genome with up to 70% efficiency of co-transformation . The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium . All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.

Arch Biochem Biophys, 1992 Jul, 296(1), 337 - 46
Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli; Imlay J et al.; The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress . However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species . Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin . Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media . An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated . The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide.

J Virol, 1992 Jul, 66(7), 4591 - 6
Activation of simian virus 40 transcription in vitro by T antigen; Coulombe J et al.; Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues . Late transcription is activated by TAg, even in the absence of viral DNA replication . We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system . In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters . When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected . Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other . Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription . An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein . Structural features of this region that might be important are discussed.

J Virol, 1992 Jul, 66(7), 4390 - 8
Expression of the Marek's disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen; Chen X et al.; Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV) . Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis . Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44 . However, the gene encoding B antigen was not unequivocally identified . Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L . J . N . Ross, M . Sanderson, S . D . Scott, M . M . Binns, T . Doel, and B . Milne, J . Gen . Virol . 70:1789-1804, 1989) . To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared . These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates . On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide . The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens . TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100 . This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above . The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection.

Rev Roum Physiol, 1992 Jul-Dec, 29(3-4), 57 - 62
Phagocytic response in rats following chemical sympathectomy with 6-hydroxydopamine; Derevenco P et al.; Wistar rats were injected i.p . at 2, 4 and 7 days after birth with 6-OHDA (50 mg/kg) . At maturity the phagocytic response of neutrophils was elicited by Escherichia coli (EC) lipopolysaccharide (LPS) . Before and at 3 and 24 hours after the injection of LPS (1 mg/kg i.v.) the leucocyte (L) and neutrophil (N) counts and the phagocytic activity of N in blood against EC have been tested . In controls (6 female and 7 male rats) the number of N increases significantly after 3 h . The L and N responses were similar in controls and in treated (SyX) animals . In controls the percentage of phagocytic active N(PA) and the number of bacteria incorporated by 100 n(IB) shows significant rises at 3 h . In the SyX groups (7 females and 7 males) PA does not increase; the IB decreases significantly in females at 3 and 24 h . In conclusion chemical sympathectomy depresses the PR to LPS in rats; this suggests a stimulatory action of the sympathetic system on the phagocytic immune reaction . An immunological sexual dimorphism exists.

Protein Sci, 1992 Jul, 1(7), 850 - 60
Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho . I . Binding of ATP; Geiselmann J et al.; Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions . We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer . This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies . We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site . The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP . The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle . The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state . This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci . 1, 861-873).

Protein Sci, 1992 Jul, 1(7), 843 - 9
Structural homology between rbs repressor and ribose binding protein implies functional similarity; Mauzy CA et al.; The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein . RbsR is also homologous to a family of bacterial repressor proteins including LacI . This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the LacI headpiece . The implications of these relationships to the mechanism of this class of repressors are discussed.

Protein Sci, 1992 Jul, 1(7), 831 - 42
Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli; Mauzy CA et al.; The DNA sequence encoding the rbs repressor protein, RbsR, has been determined . Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein . DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon . RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences.

J Postgrad Med, 1992 Jul-Sep, 38(3), 112 - 4, 111
Hyperbaric oxygen therapy in diabetic foot; Doctor N et al.; To study the effect of hyperbaric oxygen therapy in chronic diabetic foot lesions, a prospective controlled study was undertaken . Thirty diabetics with chronic foot lesions were randomised to study group (conventional management and 4 sessions of hyperbaric oxygen therapy) and control group (conventional management) . The patients were assessed for average hospital stay, control of infection and wound healing . The control of infection spread was quicker . Positive cultures decreased from initial 19 to 3 in study group as against from 16 to 12 in the control group . (p < 0.05) . This difference was most pronounced for Escherichia coli . Also, the need for major amputation was significantly less in the study group (n = 2) as against the control group (n = 7) (p < 0.05) . The average hospital stay was not affected . We conclude that hyperbaric oxygen therapy can be safely used and is beneficial as an adjuvant therapy in chronic diabetic foot lesions.

Fiziol Zh, 1992 Jul-Aug, 38(4), 56 - 62
{The ultrastructural changes in the glomeruli and juxtaglomerular apparatus at different periods of endotoxic shock}; Kharlanova NG et al.; It is shown that after endotoxin injection the ultrastructural changes in the glomeruli can favour development of the acute renal insufficiency . In the initial and intermediate periods of the endotoxin shock the granular and agranular forms of juxtaglomerular cells hyperfunction, respectively, are revealed as well as an increase of renin activity in plasma . At the stage of the late endotoxemia ultrastructural alterations are stabilized . The juxtaglomerular cells synthesize and accumulate secretory granules, but renin activity in plasma decreases almost to the initial level.

J Struct Biol, 1992 Jul-Aug, 109(1), 70 - 7
Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy; Wagenknecht T et al.; The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains) . In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC . From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions . To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin . In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core . Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro . Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core . The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3 . These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.

Protein Sci, 1992 Jul, 1(7), 861 - 73
Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho . II . Binding of RNA; Geiselmann J et al.; The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites . In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho . We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ({rC}n where n > or = 6) . Rho binds to long RNA chains as a hexamer characterized by D3 symmetry . Each hexamer binds approximately 70 residues of RNA . We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer . The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers . The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues . Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho . Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

J Neurobiol, 1992 Jul, 23(5), 481 - 90
Preparation and biological properties of native and recombinant ciliary neurotrophic factor; Gupta SK et al.; CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE) . In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons . The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed . With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves . After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons . In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 987 - 92
Identification of two types of homologous DNA pairing activity in mouse cells; Mishina Y et al.; We have identified two types of homologous DNA pairing activity in mouse cell extracts by a strand-transfer assay . Both activities are separated from each other by anion-exchange chromatography; neither of them needs ATP . One requires magnesium ion and is stimulated by Escherichia coli single-stranded DNA binding protein, whereas the other does not require the ion and shows a higher affinity for a left-handed Z-DNA.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 874 - 80
The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding; Schauer M et al.; HIV-1 integrase binds to both double- and single-stranded DNA with Kd-values of around 20 nM, irrespective of sequence similarities with the termini of the viral LTR . For integration activity, however, the correct LTR sequence of the substrate is required . The putative zinc-binding site present at the N-terminus of the protein is not essential for DNA binding, since deletion mutants of the protein lacking this sequence show similar affinity towards DNA as the wild-type; however, these mutants are not capable of performing the LTR-cleavage and integration reactions . Thus, it appears that the N-terminal part of the integrase is essential for catalytic activity.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1133 - 40
Inhibition of DNA polymerases by tripeptide derivative protease inhibitors; Taguchi T et al.; Benzyloxycarbonyl(Z)-Leu-Leu-Leu-al and dansyl(Dns)-Leu-Leu-Leu-CH2Cl, well known as protease inhibitors, effectively inhibit the activities of DNA polymerases alpha, beta and gamma from rat liver and pol I from Escherichia coli, but the ability of these inhibitors to inhibit terminal deoxynucleotidyl transferase (TdT) is weak . The mode of inhibition by these tripeptide analogues is non-competitive with dNTP . The Ki values for Z-Leu-Leu-Leu-al and Dns-Leu-Leu-Leu-CH2Cl are 6.25 x 10(-5) M and 6.56 x 10(-5) M, respectively.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1000 - 4
Proteolytic processing of amyloid beta protein precursor (APP) by thrombin; Igarashi K et al.; Search for proteases responsible for an altered processing of APP which generates intermediates containing beta/A4 peptide is preceding to understand the formation of beta amyloid deposits characteristic of Alzheimer's disease, since many studies reveal that APP is ordinarily processed so as not to generate beta amyloid . Here, we have examined the action of thrombin, a serine protease in the blood clotting, in APP processing . Thrombin cleaved the mouse recombinant APP695 in vitro, resulting in the accumulation of 28 kDa fragment . The immunoblot analysis showed that the fragment is derived from the carboxy-terminal side of the recombinant APP695 . Further, amino acid sequencing exhibited that the fragment is generated by the cleavage at Arg 510-Ile 511 and therefore includes entire beta/A4 peptide . We consider that the 28 kDa fragment is a possible intermediate for beta/A4 peptide . Thus thrombin may be involved in the altered processing of APP.

Biochemistry, 1992 Jun 30, 31(25), 5937 - 44
The mRNA binding track in the Escherichia coli ribosome for mRNAs of different sequences; Bhangu R et al.; Interactions between mRNA and rRNA on the 30S ribosomal subunit or 70S ribosome have been determined by photochemical cross-linking experiments using synthetic mRNA analogs substituted with 4-thiouridine . A set of RNA molecules containing different sequences has been used to determine the extent to which binding contacts are sequence dependent . The 16S rRNA and 23S rRNA nucleotides that form a part of the binding site have been identified by reverse transcription . The nucleotides are U1381, G1338, G1300, G1156, A845, U723, G693, A532, G497, U420, G413/A412, and G436 of 16S rRNA and U887 of 23S rRNA . Several additional nucleotides (U1065 of 23S rRNA and A1227, G818, G524, and G423 of 16S rRNA) are seen for some, but not all, of the mRNAs . Results obtained with two mRNAs containing the Shine-Dalgarno sequence were similar to those obtained with mRNAs lacking the Shine-Dalgarno sequence . Eight of these cross-linking sites were also seen when a mixture of RNA was used in which there are 12 random nucleotides preceding and seven random nucleotides succeeding an AUG codon . These results indicate that to a large extent placement of the mRNA in the ribosome does not depend upon its primary sequence.

Biochemistry, 1992 Jun 30, 31(25), 5878 - 87
Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate; Yano T et al.; Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP) . This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis . The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme . In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH . The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme . Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme . While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate . In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme . The presteady-state reactions of these two mutant enzymes with {2-2H}aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that {kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H . (1990) Biochemistry 29, 5469-5476} for the wild-type enzyme . These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1 . Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.

Biochemistry, 1992 Jun 30, 31(25), 5861 - 8
Mechanism of action of sparsomycin in protein synthesis; Theocharis DA et al.; Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI . The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition) . The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1 . The isomerized C*I, free of excess I, can be studied as a mixture with complex C . From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1 . Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity . This result does not support the proposed {Flynn, G . A., & Ash, R . J., (1990) Biochem . Biophys . Res . Commun . 166, 673-680} chemical reaction between D and I toward new products . The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M . In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction) . When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jun 30, 31(25), 5841 - 8
Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognition; Botfield MC et al.; B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins . A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2 . Octamer recognition is mediated by the POU domain, a conserved structural motif which--like the zinc finger and leucine zipper--defines a family of related transcription factors . Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence . As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin . Biochemical and biophysical characterization yields the following results . (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity . (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker . (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jun 30, 31(25), 5687 - 91
Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase; Texter FL et al.; The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation . Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation . The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR . Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization . The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process . Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond . The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1098 - 107
Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region; Presta M et al.; Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA . The bFGF mutant (M6B-bFGF) was expressed in E . coli and purified to homogeneity . When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator . In vivo, M6B-bFGF lacked a significant angiogenic activity . Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.

Biochemistry, 1992 Jun 30, 31(25), 5918 - 25
Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy; Brown RL; In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits . Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system . In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence . Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta . Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C . Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly . The results indicate that mutations in many regions of gamma affect its binding to alpha beta . A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity . However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1992 Jun 29, 305(2), 81 - 5
Heat-labile enterotoxin crystal forms with variable A/B5 orientation . Analysis of conformational flexibility; Sixma TK et al.; A new native crystal form of heat-labile enterotoxin (LT) has two AB5 complexes in the asymmetric unit with different orientations of the A subunit with respect to the B pentamer . Comparison with other crystal forms of LT shows that there is considerable conformational freedom for orientating the A subunit with respect to the B pentamer . The rotations of A in different crystal forms do not follow one specific axis, but most of them share a hinge point, close to the main interaction area between A and B5 . Analysis of the two high-resolution structures available shows that these rotations cause very little change in the actual interactions between A and B5.

J Chromatogr, 1992 Jun 26, 604(1), 39 - 46
Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers; Torres AR et al.; The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated . Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components . Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity . The demonstration of the purification of alkaline phosphatase from E . coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed . The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed . A simplified method for preparing carboxymethyldextran displacers is presented.

J Med Chem, 1992 Jun 26, 35(13), 2467 - 73
NMR studies of an FK-506 analog, {U-13C}ascomycin, bound to FK-506-binding protein; Petros AM et al.; Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for {U-13C}ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons . The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the {U-13C}ascomycin/FKBP . This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506 . However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A . The methods used for resonance assignment and structure calculation are presented in detail . Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket . This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

Science, 1992 Jun 26, 256(5065), 1783 - 90
Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor; Kohlstaedt LA et al.; A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer . The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I . However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions . Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site . The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases . Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites . Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site . Residues whose mutation results in drug resistance have been approximately located.

Mol Cell Biochem, 1992 Jun 26, 112(2), 135 - 42
Heart sarcolemmal Ca2+ transport in endotoxin shock: II . Mechanism of impairment in ATP-dependent Ca2+ transport; Liu MS et al.; The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated . The results indicate that the ATP-dependent Ca2+ transport in canine cardiac sarcolemma was decreased by 30-35% 4 h after endotoxin administration . Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca2+ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals . Dephosphorylation of sarcolemma decreased ATP-dependent Ca2+ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin . Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection . Digestion of control heart sarcolemma with phospholipase A2 inhibited Ca2+ transport and the inhibition was reversible by phosphatidylcholine . The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine . Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca2+ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.

Mol Cell Biochem, 1992 Jun 26, 112(2), 125 - 33
Heart sarcolemmal Ca2+ transport in endotoxin shock: I . Impairment of ATP-dependent Ca2+ transport; Wu LL et al.; Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated . The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p less than 0.05) at 4 h following endotoxin administration . The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p less than 0.01) and 32% (p less than 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration . Magnesium (1-5 mM) stimulated while vanadate (0.25-3.0 microM) inhibited the ATP-dependent Ca2+ transport, but the Mg(2+)-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals . These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+ . Additional experiments show that the Ca2+ sensitivity of the Ca(2+)-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca(2+)-ATPase activity . Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 687 - 95
Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex . Reversal of the endogenous feedback inhibition by phosphorylation of serine-40; Andersson KK et al.; Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days . The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component . Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate . The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1) . The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme {Andersson, Cox, Que, Flatmark & Haavik (1988) J . Biol . Chem . 263, 18621-18626} . In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit) . The e.p.r . spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes . After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r . species could be observed . Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e . high endogenous catecholamine) concentration.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3199 - 205
Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha; Takemura M et al.; We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha . The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively) . In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E . coli) . Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E . coli and liverwort chloroplast.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3179 - 82
Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity; Prestle J et al.; Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated . Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L . (pokeweed), Dianthus barbatus L . (carnation), Spinacia oleracea L . (spinach) or Chenopodium amaranthicolor Coste and Reyn . (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides . The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP . This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation . These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs . Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3127 - 33
Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis . Human glutathione reductase cDNA as a model; Bucheler US et al.; Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon . In the mTI subregion the 8 bases flanking the invariant Shine-Dalgarno motif GG-AG were mutagenized exhaustively, while the MTI subregion was subjected to random silent mutations at the wobble positions . The quality of a given TI sequence was judged on the basis of expressed enzyme activity . Low-yield and high-yield mutants of both TI subregions were selected and recombined systematically . The analysis of these double cartridges gave the following results: 1 . As a rule, an unfavourable MTI subregion can be compensated for by mutations in the mTI subregion and vice versa . 2 . The compatibility between mTI and MTI subregion is explainable at least in part by a low interaction tendency; a delta G(o)'-value of -10.7 kcal/mol appears to be a physical threshold for heterologous cDNA expression . 3 . On the basis of periShine-Dalgarno mutations, the expression yield for different cDNA sequences could be increased by 1 to 2 orders of magnitude . One of these sequences encoded delta(1-15)human glutathione reductase, a mutant lacking the flexible N-terminal extension of the protein . In conclusion, to study and overcome TI region-based expression problems it is worthwhile to start out with a versatile vector containing exhaustive mutations in the periShine-Dalgarno sequences; as a rule the coding MTI subregion can be kept unchanged.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3079 - 84
Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA; Tudek B et al.; Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua) . The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E . coli DNA polymerase I . The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA . Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base) . The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells . Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA . In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations . These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria . The results also imply that the Fapy-7-MeGua in E . coli cells is primarily a lethal lesion.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3063 - 7
Dbp73D, a Drosophila gene expressed in ovary, encodes a novel D-E-A-D box protein; Patterson LF et al.; Proteins of the D-E-A-D family of putative ATP-dependent RNA helicases have been implicated in translation initiation and RNA splicing in a variety of organisms from E . coli to man . The Drosophila vasa protein, a member of this family, is required in the female germ line for fertility and for specification of germ line and posterior positional information in progeny embryos . We report the isolation of another D-E-A-D gene from Drosophila, which, like vasa, is expressed in germ line tissue . The predicted amino acid sequence of this new gene, Dbp73D, contains all of the highly conserved helicase motifs, but is otherwise the farthest-diverged member of the family so far identified.

J Biol Chem, 1992 Jun 25, 267(18), 13062 - 72
A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4; Strack B et al.; DNA primases encoded by the conjugative plasmids ColIb-P9 (IncI1), RP4, and R751 (IncP), and the protein of the Escherichia coli satellite phage P4 alpha were shown to contain a common amino acid sequence motif -E-G-Y-A-T-A- . The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates . This priming activity resembles the enzymatic activity of DNA primases encoded by conjugative plasmids in terms of template utilization and the ability to synthesize primers that can be elongated by DNA polymerase III holoenzyme . The -E-G-Y-A-T-A- motif is part of an extended sequence region most conserved within the primase domains of the four enzymes . Single amino acid substitutions generated in the -E-G-Y-A-T-A- motif of the RP4 TraC2 and the P4 alpha protein affect priming activity, supporting the hypothesis that the conserved sequence motif is part of the active center for primase function . A mutation that eliminates priming activity causes P4 phage to grow poorly and to depend upon the host dnaG primase . Computer analysis identified two additional sequence motifs within the amino acid sequence of the P4 alpha protein: a potential zinc-finger motif and a "type A" nucleotide binding site, both strikingly similar to sequence motifs described in various DNA primases and helicases.

J Biol Chem, 1992 Jun 25, 267(18), 12986 - 90
Four conserved cysteine residues are required for the DNA binding activity of nuclear factor I; Novak A et al.; The role of Cys residues in the site-specific DNA binding activity of the nuclear factor I (NFI) family of proteins was assessed by chemical modification and site-specific mutagenesis . Treatment with the thio-specific reagent N-ethylmaleimide abolished site-specific DNA binding of all forms of NFI present in HeLa nuclear extracts . Preincubation of cell extracts with an oligonucleotide containing an NFI-binding site provided partial protection of NFI from N-ethylmaleimide inactivation . Mutations were made in the cDNA encoding a truncated form of the NFI-C/CAAT box transcription factor-1 protein, converting each of the five Cys residues in the DNA-binding domain of the protein into Ser residues . NFI-C proteins containing mutations in any of four conserved Cys residues, expressed in Escherichia coli or in vitro, did not bind to DNA . NFI-C with a mutation in a nonconserved Cys residue had normal DNA binding activity . Both this active mutant and wild-type NFI-C protein were inactivated by modification of their sulfhydryl residues with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and preincubation with an oligonucleotide containing an NFI-binding site gave partial protection against inactivation . After modification with DTNB, DNA binding activity was partially restored by subsequent incubation with dithiothreitol, indicating that inactivation of NFI by DTNB was reversible . These studies indicate an essential role for free sulfhydryl residues in NFI-DNA binding.

J Biol Chem, 1992 Jun 25, 267(18), 12885 - 91
Protein targeting across the three membranes of the Euglena chloroplast envelope; Shashidhara LS et al.; A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes . Preparation of functional chloroplasts with intact envelope membranes has been optimized . Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62% . Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors . The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa) . A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported . Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope . Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size . However, this was not imported into Euglena chloroplasts, although again it bound to them . These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence . The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.

J Biol Chem, 1992 Jun 25, 267(18), 12820 - 5
Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain; Kastern W et al.; The gene for protein L, an immunoglobulin (Ig) light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, was cloned and sequenced . The gene translates into a protein of 719 amino acid residues . Following a signal sequence of 18 amino acids and a NH2-terminal region ("A") of 79 residues, the molecule contains five homologous "B" repeats of 72-76 amino acids each . Further, toward the COOH terminus, two additional repeats ("C") were found . These are not related to the "B" repeats, but are highly homologous to each other . After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region ("W") was found, followed at the COOH-terminal end by a hydrophobic membrane anchor ("M") . Fragments of the gene were expressed, and the corresponding peptides were analyzed for Ig-binding activity . The B repeats were found to be responsible for the interaction with Ig light chains . An Escherichia coli high level expression system was adapted for the production of large amounts of two Ig-binding protein L fragments comprising one and four B repeats, respectively.

J Biol Chem, 1992 Jun 25, 267(18), 12655 - 9
Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen; Gertler A et al.; Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation . However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity . The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity . Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells) . Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure . Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL . Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties . It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors . Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.

J Biol Chem, 1992 Jun 25, 267(18), 12627 - 31
Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase; Larsen IK et al.; The anticancer drug caracemide, N-acetyl-N,O- di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1 . No effect on the smaller protein R2 was observed . The effect of the degradation product was about 30 times lower than that of caracemide itself . The caracemide inactivation of R1 is irreversible, with an apparent second-order rate constant of 150 M-1 s-1 . The R1R2 holoenzyme was approximately 30 times more sensitive to caracemide inactivation than the isolated R1 protein . The ribonucleotide reductase substrates were potent competitors of the caracemide inhibition, with a Kdiss for GDP binding to R1 of 80 microM . The reducing agent dithiothreitol was also found to be a potent competitor of caracemide inactivation . These results indicate that caracemide inactivates R1 by covalent modification at the substrate-binding site . By analogy with the known interaction between caracemide and acetylcholinesterase or choline acetyltransferase, we propose that the modification of R1 occurs at an activated cysteine or serine residue in the active site of the enzyme.

J Biol Chem, 1992 Jun 25, 267(18), 12622 - 6
Escherichia coli ribonucleotide reductase . Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme; Karlsson M et al.; Ribonucleotide reductase catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides via a radical-mediated mechanism . The enzyme from Escherichia coli consists of the two non-identical proteins, R1 and R2, the latter of which contains the necessary free radical located to a tyrosine residue . The radical scavenger hydroxyurea was found to reduce the tyrosyl radical of R2 in a second-order reaction . The rate constant (0.50 M-1 s-1 at 25 degrees C) for this process was several orders of magnitude lower than the hydroxyurea-dependent reduction of free tyrosyl radicals in solution . This difference probably reflects the fact that the R2 tyrosyl radical is buried in the interior of the protein . Formation of the R1R2 complex changed the susceptibility of the radical to hydroxyurea in a manner that reflects the regulatory state of the holoenzyme . Furthermore, binding of substrate or product to the holoenzyme complex made the R2 radical at least 10 times more susceptible to inactivation by hydroxyurea than it was in the isolated R2 protein . One active site mutation in the R1 protein was shown to affect the sensitivity of the tyrosyl radical of R2 differently than wild type protein R1 does . Our results clearly show that the susceptibility of the tyrosyl radical in R2 to inactivation by hydroxyurea can be used as an efficient probe for the regulatory state of the holoenzyme complex.

J Biol Chem, 1992 Jun 25, 267(18), 12860 - 7
Oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase . The carboxyl-terminal region of OSCP is essential for the reconstitution of oligomycin-sensitive H(+)-ATPase; Joshi S et al.; Studies to establish the structure/function relationships of oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase were carried out using genetic engineering and biochemical approaches . A full-length cDNA clone encoding OSCP was isolated from a bovine heart cDNA library, and the mature form of OSCP was expressed in Escherichia coli using plasmid expression vector pKP1500 . Recombinant OSCP was found to accumulate in the cytoplasmic inclusion bodies, by virtue of which the recombinant protein could be purified to greater than 85% purity by simple low speed centrifugation of cell lysates . Recombinant OSCP was found to be indistinguishable from OSCP isolated from mitochondria with respect to (i) apparent molecular mass on sodium dodecyl sulfate gel electrophoresis, (ii) immunological reactivity to anti-OSCP serum, (iii) biological activity in restoring oligomycin-sensitive ATPase and Pi-ATP exchange activities to OSCP-depleted ATP synthase complexes, and (iv) insensitivity of the biological activity to sulfhydryl-directed alkylating reagents . The amino-terminal sequence of the recombinant protein revealed that the initiating methionine was not removed by E . coli, although that apparently did not affect protein folding or its biological activity . Data on nested deletion mutations starting from the carboxyl terminus in OSCP demonstrated that, in each instance, the mutant form was expressed and the protein product was sequestered in cytoplasmic inclusion bodies, similar to the wild-type form . However, none of the variants, including the one in which only the last 10 residues were deleted, was able to restore cold-stable oligomycin-sensitive ATPase or Pi-ATP exchange activity in OSCP-depleted complexes . Taken together, these data suggest that amino acid residues 181-190 (or some of the residues in this region) in the OSCP sequence may be important for OSCP-F1 interactions.

J Biol Chem, 1992 Jun 25, 267(18), 12717 - 21
Rapid, high yield purification and characterization of the K(+)-translocating Kdp-ATPase from Escherichia coli; Siebers A et al.; The conventional procedure for the purification of the high affinity K+ uptake ATPase (KdpABC) from Escherichia coli involves a tedious three-column protocol (final enzyme purity, approximately 90%; activity yield, 6.5% (Siebers, A., and Altendorf, K . (1988) Eur . J . Biochem . 178, 131-140)) . We have now developed a highly effective one-column (Fractogel TSK AF-Red) protocol yielding an enzyme preparation of comparable purity with severalfold higher activity yield . A further increase in enzyme purity up to 98% was achieved by a two-column protocol involving elution over DEAE-Sepharose CL-6B prior to TSK AF-Red affinity chromatography . The reduction of preparation time minimized KdpB protein degradation and led to hitherto unequaled values of specific activity (up to 2000 mumols x g-1 x min-1) and enrichment factors (up to 30-fold) . Our results confirm the usefulness of triazine dye matrices for the purification of transport ATPases.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3233 - 40
Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer; Yoshimura K et al.; As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice . To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice . With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk . With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice . Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation . Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E . coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene . These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo . This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3147 - 52
Novel mutants of 23S RNA: characterization of functional properties; Saarma U et al.; Single point mutations corresponding to the positions G2505 and G2583 have been constructed in the gene encoding E.coli 23S rRNA . These mutations were linked to the second mutation A1067 to T, known to confer resistance to thiostrepton (1) . Mutant ribosomes were analyzed in vitro for their ability to direct poly(U) dependent translation, their missence error frequency and in addition their sensitivity to peptidyltransferase inhibitors . It was evident that the mutated ribosomes had an altered dependence on {Mg2+} and an increased sensitivity to chloramphenicol during poly(U) directed poly(Phe) synthesis . In a transpeptidation assay mutated ribosomes were as sensitive to chloramphenicol as wild-type ribosomes . However, the mutant ribosomes exhibited an increased sensitivity to lincomycin . An increase in translational accuracy was attributed to the mutations at the position 2583: accuracy increased in the order G less than A less than U less than C.

J Biol Chem, 1992 Jun 25, 267(18), 12742 - 52
Phosphorylation of the cystic fibrosis transmembrane conductance regulator; Picciotto MR et al.; Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC) . Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli . CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I) . Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC . CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2 . Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG . CFTR was immunoprecipitated from T84 cells labeled with 32Pi . Its phosphorylation was stimulated in response to agents that activated either PKA or PKC . Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro . Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.

J Biol Chem, 1992 Jun 25, 267(18), 12639 - 46
Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase . Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity; Daubner SC et al.; Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P . F., Chlumsky, L . J., Daubner, S . C., and O'Malley, K . L . (1990) J . Biol . Chem . 265, 2042-2047) . The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of {32P}phosphate . Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7 . Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate . Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7 . Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold . Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold . The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells . In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions . These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.

J Biol Chem, 1992 Jun 25, 267(18), 12400 - 3
Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli; Wynn RM et al.; We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli . The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains . Only small or trace amounts of active E1 component were obtained . Cotransformation of the ES- mutant host with a second vector overexpressing groEL and groES resulted in a greater than 500-fold increase in E1-specific activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the content of both MBP-E1 alpha and E1 beta polypeptides was markedly increased in the presence of overexpressed chaperonin proteins . The time course studies showed that the increase in E1-specific activity and subunit levels correlated with the increase in groEL and groES until the concentration of the chaperonins reached a saturating level in the cell . The functional MBP-E1 fusion protein from ES- double transformants were purified by amylose resin affinity chromatography . The MBP moiety was removed by subsequent digestion with Factor Xa endoprotease, followed by Sephacryl S-300HR chromatography . It was found that E1 alpha and E1 beta assembled into an active 160-kDa species, which was consistent with the alpha 2 beta 2 structure of E1 . The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3159 - 65
Cloning and characterization of the mvrC gene of Escherichia coli K-12 which confers resistance against methyl viologen toxicity; Morimyo M et al.; A new gene mvrC conferring resistance to methyl viologen, a powerful superoxide radical propagator, was cloned on 13.5 kilo base (kb) EcoRI DNA fragment . It gave resistance against methyl viologen to even a wild-type strain with gene dosage dependence . From the physical maps obtained by restriction enzyme digestions, it was predicted to locate at 580 kbp (12.3 min) on the physical map of E.coli . This was confirmed by the Southern hybridization of lambda phages covering this region with mvrC probe . The DNA sequence of mvrC gene was determined and its deduced protein encoding a 12 kd hydrophobic protein was confirmed by maxicell labeling of MvrC protein.

J Biol Chem, 1992 Jun 25, 267(18), 12813 - 9
Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage; Severinov K et al.; The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G . C., Lim, P.-P., Glass, R . E., Nikiforov, V., and Goldfarb, A . (1991) J . Biol . Chem . 266, 23921-23926) . Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin . We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity . Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.

Biochim Biophys Acta, 1992 Jun 24, 1121(3), 286 - 92
Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese; Miller DM et al.; Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography . Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000 . The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E . coli also removes the N-terminal methionine . The Km for thiosulfate is the same for the two proteins . The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg) . The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence . The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E . coli chaperonin system composed of cpn60 and cpn10 . Bovine rhodanese is known to have multiple electrophoretic forms under native conditions . In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms . This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

Biochim Biophys Acta, 1992 Jun 24, 1121(3), 293 - 6
Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes; Haberlein I et al.; Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin . The modified thioredoxins alone are inactive . These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.

Biochemistry, 1992 Jun 23, 31(24), 5553 - 60
A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor; Read BA et al.; Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp . N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301 . The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E . coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes . Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively . Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP . The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate . Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO . Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins . These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.

Biochemistry, 1992 Jun 23, 31(24), 5528 - 34
EPSP synthase: binding studies using isothermal titration microcalorimetry and equilibrium dialysis and their implications for ligand recognition and kinetic mechanism; Ream JE et al.; Isothermal titration calorimetry measurements are reported which give important new binding constant (Kd) information for various substrate and inhibitor complexes of Escherichia coli EPSP synthase (EPSPS) . The validity of this technique was first verified by determining Kd's for the known binary complex with the substrate, shikimate 3-phosphate (S3P), as well as the herbicidal ternary complex with S3P and glyphosate (EPSPS.S3P.glyphosate) . The observed Kd's agreed very well with those from previous independently determined kinetic and fluorescence binding measurements . Further applications unequivocally demonstrate for the first time a fairly tight interaction between phosphoenolpyruvate (PEP) and free enzyme (Kd = 390 microM) as well as a correspondingly weak affinity for glyphosate (Kd = 12 mM) alone with enzyme . The formation of the EPSPS.PEP binary complex was independently corroborated using equilibrium dialysis . These results strongly suggest that S3P synergizes glyphosate binding much more effectively than it does PEP binding . These observations add important new evidence to support the hypothesis that glyphosate acts as a transition-state analogue of PEP . However, the formation of a catalytically productive PEP binary complex is inconsistent with the previously reported compulsory binding order process required for catalysis and has led to new studies which completely revise the overall EPSPS kinetic mechanism . A previously postulated ternary complex between S3P and inorganic phosphate (EPSPS.S3P.Pi, Kd = 4 mM) was also detected for the first time . Quantitative binding enthalpies and entropies were also determined for each ligand complex from the microcalorimetry data . These values demonstrate a clear difference in thermodynamic parameters for recognition at the S3P site versus those observed for the PEP, Pi, and glyphosate sites.

Biochemistry, 1992 Jun 23, 31(24), 5514 - 21
Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli; Lolkema JS et al.; The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr . The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique . On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically . One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume . The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol . Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane . Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.

Biochemistry, 1992 Jun 23, 31(24), 5449 - 58
Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis . 1 . Crystallography and site-directed mutagenesis of metal binding sites; Jenkins J et al.; The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli . The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution . The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors . Two metal sites are identified . Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1 . Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein . Side chains involved in metal binding have been substituted by site-directed mutagenesis . The biochemical properties of the mutant enzymes are presented . Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

Biochem Pharmacol, 1992 Jun 23, 43(12), 2581 - 9
Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers; Austermann S et al.; Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha {pJS3.7} . Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate {poly(rA).(dT)20} and polyribouridylate 20-mer of (3'-5')-deoxyadenylate {poly(rU).(dA)20} homopolymeric template-primers . Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates . Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively . The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured . Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT . In particular, poly(rA).(dT)20-{ddTMP} and poly(rU).(dA)20-{3'-dAMP} were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively . Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM . In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM . Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM . Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT . This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.

Biochemistry, 1992 Jun 23, 31(24), 5423 - 9
Recognition of G-U mismatches by tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III); Chow CS et al.; The coordination complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) {Rh(DIP)3(3+)}, which promotes RNA cleavage upon photoactivation, has been shown to target specifically guanine-uracil (G-U) mismatches in double-helical regions of folded RNAs . Photoactivated cleavage by Rh(DIP)3(3+) has been examined on a series of RNAs that contain G-U mismatches, yeast tRNA(Phe) and yeast tRNA(Asp), as well as on 5S rRNAs from Xenopus oocytes and Escherichia coli . In addition, a "microhelix" was synthesized, which consists of seven base pairs of the acceptor stem of yeast tRNA(Phe) connected by a six-nucleotide loop and contains a mismatch involving residues G4 and U69 . A U4.G69 variant of this sequence was also constructed, and cleavage by Rh(DIP)3(3+) was examined . In each of these cases, specific cleavage is observed at the residue which lies to the 3'-side of the wobble-paired U; some cleavage by the rhodium complex is also evident in several structured RNA loops . The remarkable site selectivity for G-U mismatches within double-helical regions is attributed to shape-selective binding by the rhodium complex . This binding furthermore depends upon the orientation of the G-U mismatch, which produces different stacking interactions between the G-U base pair with the Watson-Crick base pair following it on the 5'-side of U compared to the Watson-Crick pair preceding it on the 3'-side of U . Rh(DIP)3(3+) therefore serves as a unique probe of G-U mismatches and may be useful both as a model and in probing RNA-protein interactions as well as in identifying G-U mismatches within double-helical regions of folded RNAs.

Biochemistry, 1992 Jun 23, 31(24), 5661 - 4
Folding and assembly of the Escherichia coli succinyl-CoA synthetase heterotetramer without participation of molecular chaperones; Fong G et al.; Succinyl-CoA synthetase of Escherichia coli (alpha 2B2 subunit structure) has been shown to fold and assemble without participation by molecular chaperones . Renaturation experiments showed that purified bacterial chaperone GroEL has no effect on the folding and assembly of the active tetrameric enzyme . When isolated 35S-labeled alpha or beta subunits were incubated with GroEL in the absence of ATP, there was no complex formation between the subunits and GroEL . These in vitro results were confirmed by in vivo analysis of the folding and assembly of newly synthesized succinyl-CoA synthetase subunits . When expression of the subunits was induced in E . coli strains that bear GroEL or GroES temperature-sensitive mutations, the assembly of active succinyl-CoA synthetase was not affected as the temperature was raised to 43 degrees C . These and other observations are discussed that indicate that folding and assembly of succinyl-CoA synthetase may be independent of assistance by any chaperone.

Biochemistry, 1992 Jun 23, 31(24), 5534 - 44
Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli; Gruys KJ et al.; Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP) . Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme . Steady-state kinetic and spectroscopic studies presented here indicate that E . coli EPSPS does indeed follow a random kinetic mechanism . Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern . Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site {Ki(PEP) = 6-8 mM} . To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates . The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM) . Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position . Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat . Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex . The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP . Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR . The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors . The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover . Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.

Biochim Biophys Acta, 1992 Jun 22, 1126(2), 206 - 14
Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme; Raghupathi R et al.; Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids . Thus, CUFAs may function as endogenous suppressors of lipolysis . To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom . Arachidonate inhibited hydrolysis of both {1-14C}oleate-labelled, autoclaved Escherichia coli and {1-14C}linoleate-labelled phosphatidylethanolamine in an apparent competitive manner . When subjected to gel permeation chromatography, {3H}arachidonate, but not {3H}palmitate, comigrated with the enzyme . Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect . Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control) . Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E . coli substrate . Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction . Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.

Proc R Soc Lond B Biol Sci, 1992 Jun 22, 248(1323), 247 - 53
Interaction of avidin with the lipoyl domains in the pyruvate dehydrogenase multienzyme complex: three-dimensional location and similarity to biotinyl domains in carboxylases; Hale G et al.; Avidin can form intermolecular cross-links between particles of the pyruvate dehydrogenase multienzyme complex from various sources . Avidin does this by binding to lipoic acid-containing regions of the dihydrolipoamide acetyltransferase polypeptide chains that comprise the structural core of the complex . It is inferred that the lipoyl domains of the acetyltransferase chain extend outwards from the interior of the enzyme particle, interdigitating between the subunits of the other two enzymes bound peripherally in the assembled structure, with the lipoyl-lysine residues capable of reaching to within at least 1-2 nm of the outer surface of the enzyme complex (diameter ca . 37 nm) . The distribution of enzymic activities between different domains of the dihydrolipoamide acetyltransferase chain implies that considerable movement of the lipoyl domains is a feature of the catalytic activity of the enzyme complex . There is evidence that the lipoyl domain of the 2-oxo acid dehydrogenase complexes is similar in structure to a domain that binds the cofactor biotin, also in amide linkage with a specific lysine residue, in the biotin-dependent class of carboxylases.

J Mol Biol, 1992 Jun 20, 225(4), 1137 - 41
Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase; Naismith JH et al.; We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli . Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis . The space group has been identified as P6(1)22 or enantiomorph . Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S . Daresbury . Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam . A data set to this resolution has been collected . Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.

J Mol Biol, 1992 Jun 20, 225(4), 1131 - 3
Crystallization and preliminary X-ray studies on the co-repressor binding domain of the Escherichia coli purine repressor; Schumacher MA et al.; The purine repressor is a putative helix-turn-helix DNA-binding protein that regulates several genetic loci important in purine and pyrimidine metabolism in Escherichia coli . The protein is composed of two domains, an N-terminal DNA-binding domain and a C-terminal core that binds the purine co-repressors, guanine and hypoxanthine . The co-repressor binding domain (residues 53 to 341) has been crystallized from polyethylene glycol 600-MgCl2 solutions . They are of the monoclinic form, space group P2(1), with a = 38.2 A, b = 125.7 A, c = 61.8 A and beta = 100.2 degrees . They diffract to a resolution of at least 2.2 A and contain two monomers per asymmetric unit . The importance of the structural determination of this domain is underscored by the high degree of sequence homology displayed within the effector binding sites among a sub-class of helix-turn-helix proteins, of which LacI and GalR are members . The structure of the PurR co-repressor binding domain will provide a high resolution view of one such domain and could serve as a possible model for future effector site structural determinations . Perhaps more important will be this structure's contribution to the further understanding of how protein-DNA interactions are modulated.

J Mol Biol, 1992 Jun 20, 225(4), 1013 - 25
Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking; Nassal M et al.; The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus . Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle . To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine . A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region . Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization . Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface . Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues . Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds . The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities . The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.

Science, 1992 Jun 19, 256(5064), 1681 - 4
Triple helix-specific ligands; Mergny JL et al.; A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA . A benzo{e}pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex . Energy transfer experiments suggest that BePI intercalates within the triple helix . Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI . Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

Biochemistry, 1992 Jun 16, 31(23), 5303 - 11
A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain; Fritsch M et al.; Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes . In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects . The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating . Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity . The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity . These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min . The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain . Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor . The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 869 - 76
Duck liver 'malic' enzyme . Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants; Hsu RY et al.; A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene . C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique . The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver . The natural duck enzyme has a subunit molecular mass of approx . 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat . at 8 mM-L-malate) . Properties of the E . coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme . Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction . The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e . inhibition by L-malate, Ka'); however, kcat . decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency {kcat./(Km NADP+ x Km L-malate x Ka)} equal to 3.7% of the natural duck enzyme . These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 827 - 34
Some properties of murine selenocysteine synthase; Mizutani T et al.; Selenocysteine (Scy) was synthesized on natural opal suppressor tRNA(Ser) by conversion from seryl-tRNA . We studied the mechanisms of the synthesis of mammalian Scy-tRNA using hydro{75Se}selenide (H75Se-) . We found Scy synthase activity in the 105,000 g supernatant of a murine liver extract . The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at 0.12 M-KCl . The reaction mixture for synthesis of Scy-tRNA contained suppressor tRNA, serine, ATP, seryl-tRNA synthetase (SerRS), HSe- and the enzyme to synthesize Scy-tRNA . These are all essential for the synthesis of Scy-tRNA . Scy in the tRNA product was confirmed by five t.l.c . systems . The conversion from seryl-tRNA to Scy-tRNA was also confirmed with the use of {14C}- and {3H}-serine . The apparent Km values for the substrates serine, tRNA, ATP and HSe- were 30 microM, 140 nM, 2 mM and 40 nM respectively . The active eluates from DEAE-cellulose contained no tRNA kinase . This result showed that Scy-tRNA was not synthesized through phosphoseryl-tRNA . ATP was necessary when Scy-tRNA was synthesized from seryl-tRNA and HSe- . Therefore ATP is used for not only the synthesis of seryl-tRNA but also for the synthesis of Scy-tRNA from seryl-tRNA . The active fraction from DEAE-cellulose was chromatographed on Sephacryl S-300, but the activity disappeared . However, the activity was recovered by mixing the eluates corresponding to proteins of 500 kDa and 20 kDa . In order to examine the binding of HSe- to proteins, a mixture of the active fraction, H75Se- and ATP was analysed by chromatography on Sephacryl S-300 . The 75Se radioactivity was found at the position of a 20 kDa protein in the presence of ATP . Thus the 20 kDa protein plays a role in binding HSe- in the presence of ATP . The 500 kDa protein must have a role in the synthesis of Scy-tRNA . There are two natural suppressor serine tRNAs, tRNA(NCA) and tRNA(CmCA), in cell cytosol . The present paper shows that the suppressor tRNA fraction, eluted later on benzoylated DEAE-(BD-)cellulose, is a better substrate with which to synthesize Scy-tRNA . Thus we consider that murine Scy-tRNA is synthesized from a suppressor seryl-tRNA on the 500 kDa protein with the activated HSe-, which is synthesized with ATP on the 20 kDa protein . This mammalian mechanism used to synthesize Scy is similar to that seen in Escherichia coli.

FEBS Lett, 1992 Jun 15, 304(2-3), 201 - 6
The identification and characterisation of an actin-binding site in alpha-actinin by mutagenesis; Kuhlman PA et al.; We have shown previously that the N-terminal actin-binding domain of alpha-actinin retains activity when expressed in E . coli as a fusion protein with glutathione-S-transferase . In the present study we have made a series of N- and C-terminal deletions within this domain and show that an actin-binding site is contained within residues 120-134 . Amino acid substitutions within this region indicate that several highly conserved hydrophobic residues are involved in binding to F-actin . The hypothesis that the interaction between alpha-actinin and F-actin is predominantly hydrophobic in nature is supported by the observation that binding is relatively independent of salt concentration.

Gene, 1992 Jun 15, 115(1-2), 43 - 8
Gene expression in the Streptomyces temperate phage phi C31; Smith MC et al.; The repressor gene, c, of the temperate Streptomyces phage, phi C31 was previously cloned and sequenced, and predicted to encode a 74-kDa protein . The c gene actually produces three in-frame, N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa . The repressor proteins are translated from a corresponding nest of transcripts . Genetic and biochemical evidence suggests that the transcription of the c locus is autoregulated possibly by the 42-kDa protein binding to a highly conserved 16-bp perfect inverted repeat . The 16-bp sequence is present at at least twelve loci throughout the phi C31 genome . Transcription of the 'early' region is complex, possibly involving phage-specific promoters . The phi C31 terminators display sequence conservation and may be regulated . The phi C31 gene 'k' may encode a nucleotide kinase-encoding gene.

Gene, 1992 Jun 15, 115(1-2), 13 - 7
Glutamine synthesis in Streptomyces--a review; Fisher SH; The synthesis of glutamine synthetase (GS), a key enzyme in ammonium (NH4+) assimilation, is regulated by nitrogen availability in several Streptomyces strains . In addition, the enzymatic activity of the GS enzyme is post-translationally regulated by adenylylation . Nitrogen regulation of GS synthesis is mediated at the transcriptional level in S . coelicolor, and transcription of the GS structural gene (glnA) requires a positive regulatory protein, GlnR . The amino acid sequence of the GlnR protein is similar to that of the Escherichia coli positive regulatory proteins, OmpR and PhoB, which belong to the family of bacterial two-component regulatory systems . DNA encoding a GSII-like enzyme has been cloned from S . viridochromogenes and S . hygroscopicus, but the role of this GS isoenzyme in NH4+ assimilation in Streptomyces is unclear.

Biochim Biophys Acta, 1992 Jun 15, 1131(2), 199 - 202
Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism; Chen JC et al.; We reported earlier the identification of two Azotobacter vinelandii open reading frames (ORFs), ORF1 and ORF2, downstream from the hydrogenase structural genes (Chen, J.C . and Mortenson, L.E . (1992) Biochim . Biophys . Acta 1131, 122-124) . Sequencing of 6008 base pairs of DNA immediately downstream from ORF2 revealed six additional ORFs (ORF3 through ORF8) . All six ORFs are transcribed from the same DNA strand as that of the ORF1 and ORF2 . Deduced amino acid sequences of ORF3 through ORF5, and those of ORF4, ORF5, ORF7 and ORF8 have strong homology with genes required for dihydrogen (H2) metabolism in Rhodobacter capsulatus and in Escherichia coli, respectively . ORF4, ORF5, ORF6 and ORF8 would encode for polypeptides containing one or more 'Cys-X-X-Cys' motifs . The predicted products of ORF5 and ORF6 each contain a histidine-rich region, and the product of ORF5 also includes a 'Cys-Thr-Val-Cys-Gly-Cys' region near its amino-terminus . Implications of these findings with respect to metal binding, transport and incorporation, to hydrogenase assembly and to H2 metabolism are discussed.

Biochim Biophys Acta, 1992 Jun 15, 1131(2), 161 - 5
Molecular cloning and expression in Escherichia coli of a cDNA clone encoding luciferase of a firefly, Luciola lateralis; Tatsumi H et al.; We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A)+ RNA, using a cDNA of L . cruciata (another common firefly in Japan) luciferase as a probe . The primary structure of L . lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60,132 . Sequence comparison indicates that L . lateralis luciferase has significant sequence identity (94%) to L . cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase . The isolated cDNA clone, when introduced into Escherichia coli, directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5680 - 4
Eight base changes are sufficient to convert a leucine-inserting tRNA into a serine-inserting tRNA; Normanly J et al.; Each aminoacyl-tRNA synthetase must functionally distinguish its cognate tRNAs from all others . We have determined the minimum number of changes required to transform a leucine amber suppressor tRNA to serine identity . Eight changes are required . These are located in the acceptor stem and in the D stem.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5577 - 81
Intervening sequences in an Archaea DNA polymerase gene; Perler FB et al.; The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli . It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons . To our knowledge, neither IVS is similar to previously described introns . However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns . The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted . IVS2 self-splices in E . coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted . Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing . These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5527 - 31
Discrimination of DNA response elements for thyroid hormone and estrogen is dependent on dimerization of receptor DNA binding domains; Hirst MA et al.; We and others have previously shown that a two-amino acid substitution in the base of the first zinc finger of the glucocorticoid receptor DNA binding domain (DBD) is sufficient to alter the receptor's target DNA from a glucocorticoid response element (GRE) to an estrogen response element (ERE) . Activation of a thyroid hormone response element (TRE) has been shown to require an additional five-amino acid change in the second zinc finger of the thyroid hormone receptor (TR) . Using closely related TRE and ERE sequences, we report that a receptor containing the TR DBD activates the ERE poorly, and receptors containing essential amino acids of the estrogen receptor (ER) DBD activate the TRE poorly . The ER DBD (expressed in Escherichia coli) selectively bound to a 32P-labeled ERE (32P-ERE) as a dimer and a 32P-TRE as a monomer, whereas the TR DBD bound 32P-TRE as a dimer and 32P-ERE as a monomer . When hybrid receptor DBDs were examined, we found that the five amino acids in the second zinc finger of the TR necessary for TRE activation were also essential for dimer formation on a TRE . Dimer formation of ER on an ERE was localized to the second half of the second zinc finger . These results suggest that the ability of ER and TR to functionally discriminate between an ERE and a TRE is a result of dimerization of their DBDs.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5452 - 6
Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions; Parsons CA et al.; The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage . In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage . In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions . Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes . Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures . The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min) . The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-{gamma-thio}triphosphate . We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E . coli.

Eur J Biochem, 1992 Jun 15, 206(3), 919 - 25
Requirement of N- and C-terminal regions for enzymatic activity of human T-cell leukemia virus type I protease; Hayakawa T et al.; The requirement of N- and C-terminal regions for the enzymatic activity of human T-cell leukemia virus type I (HTLV-I) protease was investigated using a series of deletion mutants . The activity was analyzed by autoprocessing of the protease itself or by processing of the gag p53 precursor . The deletional analyses indicated that Asp38-Gly152 with an additional Met-Pro sequence at the N-terminus was probably sufficient for the enzymatic activity, although the mature HTLV-I protease consists of Pro33-Leu157 . A molecular model of HTLV-I protease, which was constructed by comparison with the structure of Rous sarcoma virus protease, predicted that Pro33-Leu37 and Gly143-Leu147 would form a beta-sheet . Our experimental results and the model structure suggest that (a) five amino acids in the N-terminal region (Pro33-Leu37), which are thought to be involved in the beta-sheet, are not crucial for the enzymatic activity; (b) Pro153-Leu157 is not necessary but Pro148-Gly152 is important for the enzymatic activity, in addition to Gly143-Leu147 involved in the beta-sheet.

Eur J Biochem, 1992 Jun 15, 206(3), 793 - 800
The pro-region of the yeast prepro-alpha-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo; Chaudhuri B et al.; Four yeast secretion signals, the 19-amino-acid invertase signal sequence, the 17-amino-acid acid-phosphatase signal sequence, and the pre-sequence and prepro-sequence of prepro-alpha-factor have been used to look for the secretion of recombinant human insulin-like growth factor 1 (IGF1) from Saccharomyces cerevisiae . Only the prepro-sequence, often referred to as the alpha-factor leader and consisting of an N-terminal 19-amino-acid pre-sequence or signal sequence attached to a 66-amino-acid pro-region, permits secretion of IGF1 . The signal sequences alone do not allow the translocation of IGF1 into the endoplasmic reticulum . This is evident from the fact that IGF1-like molecules, to which the signal sequences are still attached, accumulate intracellularly in the cytosol . Fusion of the pro-region of the alpha-factor leader to the C-terminus of the acid-phosphatase and invertase signal sequences allows IGF1 to be secreted once again . These results reveal the essential role of the pro-region of the alpha-factor leader in the secretion of IGF1 and indicate that it may have a function in guiding a nascent IGF1 polypeptide to a state in which translocation can occur.

J Biol Chem, 1992 Jun 15, 267(17), 12182 - 7
Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein . Identification of essential domains for nuclear localization and DNA excision repair; Miyamoto I et al.; We showed previously that the xeroderma pigmentosum group A complementing (XPAC) protein involved in the DNA excision repair pathway contains a zinc-finger motif and is localized in the nucleus of normal human cells . For detailed structural and functional analyses of the XPAC protein, we constructed various XPAC cDNAs by site-directed mutagenesis and isolated permanent cell lines expressing mutant proteins . Immunofluorescent analysis of these lines indicated that the nuclear localization signal is located in the region encoded by Exon 1, especially centered at amino acids 30-42 . A UV survival study showed that regions from Exons 2 through 6 were essential for DNA repair function, but that Exon 1 was not . Interestingly, deletion of the glutamic acid cluster in the region encoded by Exon 2 resulted in a dramatic loss of DNA repair activity . Furthermore, replacements of each of the 4 cysteines supposed to form a zinc-finger structure in the region encoded by Exon 3 by serine or glycine resulted in similar levels of loss of repair activity . These results suggest that all 4 cysteines forming a zinc-finger structure and also the glutamic acid cluster are important for DNA repair function.

J Biol Chem, 1992 Jun 15, 267(17), 12142 - 8
Initiation of methyl-directed mismatch repair; Au KG et al.; Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K . M., Lu, A.-L., Clark, S., and Modrich, P . (1987) J . Biol . Chem . 262, 15624-15629) . MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA . The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation . Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC) . Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine . The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP . The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand . However, activation is sensitive to DNA topology . Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule . MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate . We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.

J Biol Chem, 1992 Jun 15, 267(17), 11972 - 6
Hyperprocessing of tRNA by the catalytic RNA of RNase P . Cleavage of a natural tRNA within the mature tRNA sequence and evidence for an altered conformation of the substrate tRNA; Kikuchi Y et al.; In the transposon copia-related retrovirus-like particles of Drosophila, a 39-nucleotide-long fragment from the 5'-region of Drosophila initiator methionine tRNA (tRNA(iMet)) is used as the primer for copia minus-strand reverse transcription . This primer tRNA(iMet) fragment is thought to be produced by cleavage within the mature tRNA(iMet) sequence . We call this cleavage hyperprocessing . We have previously reported that catalytic RNA of RNase P from Escherichia coli (M1RNA) cleaves the synthetic tRNA(iMet) precursor in vitro at several sites within the mature tRNA sequence . Based on this result, we proposed a model for formation of the primer tRNA fragment involving RNase P . Here we show that natural tRNA(iMet) prepared from Drosophila adult flies can be cleaved by M1RNA . Using mutant tRNA(iMet) substrates, we also show that these cleavages are dependent on the occurrence of an altered conformation of the tRNA substrate . This is evidence that a tRNA can exist in aqueous solution at least in part in an altered conformation.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5226 - 30
RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot; Taylor AF et al.; During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli . We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites . Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected . These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.

J Biol Chem, 1992 Jun 15, 267(17), 12350 - 5
Overexpression and purification of ferric enterobactin esterase from Escherichia coli . Demonstration of enzymatic hydrolysis of enterobactin and its iron complex; Brickman TJ et al.; The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein . The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography . Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages . Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand . It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal . Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated . However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes . These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E . coli.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 841 - 5
Epitope mapping reveals conserved regions of an auxin-binding protein; Napier RM et al.; There is now good evidence that maize (Zea mays) auxin-binding protein (ABP) functions as a receptor . We have synthesized sequential overlapping hexapeptides to map the epitopes recognized by a number of antisera to ABP . Only a few regions of the protein are recognized, and these are shown to be exposed on the surface . Three epitopes predominate, and these are clustered around, but do not include, the glycosylation site . A comparison is made between these maps of sera against purified ABP, maps of sera raised against recombinant maize ABP expressed in Escherichia coli and computer antigenicity predictions . Our anti-(maize ABP) serum recognizes ABP counterparts in other plant species . We have used immunoblotting to affinity-purify the immunoglobulins which cross-react from the antiserum . Epitope mapping of these immunoglobulins suggests that two of the three predominant epitopes may be conserved in both monocotyledonous and dicotyledonous plants . The possible functional significance of these conserved epitopes is discussed.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 711 - 5
Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa; Pietu G et al.; In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9) . This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment {amino acids (aa) 1366-2050}, which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) . A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase . Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones . vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9 . vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting . In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM . Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5398 - 402
A family of concanavalin A-binding peptides from a hexapeptide epitope library; Scott JK et al.; The lectin concanavalin A (Con A) binds methyl alpha-D-mannopyranoside (Me alpha Man) as well as alpha-D-mannosyl groups at the nonreducing terminus of oligosaccharides . Ligand peptides that mimic the binding of Me alpha Man to Con A were identified from screening an epitope library composed of filamentous phage displaying random hexapeptides . A consensus sequence was identified among affinity-purified phage; Con A binds phage bearing this sequence and is inhibited from doing so by Me alpha Man . When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective . A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl alpha-D-glucopyranoside . These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.

J Biol Chem, 1992 Jun 15, 267(17), 12370 - 4
Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli . II . Effects of subunit c-specific polyclonal antibodies; Deckers-Hebestreit G et al.; After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked . Rebinding of F1 to those vesicles is affected by the antibody concentration used . In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results . However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1 . This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000 . Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked . Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments . Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.

J Biol Chem, 1992 Jun 15, 267(17), 12364 - 9
Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli . I . Effects of subunit b-specific polyclonal antibodies; Deckers-Hebestreit G et al.; Incubation of F1-stripped everted membrane vesicles with antibodies against subunit b of the ATP synthase from Escherichia coli resulted in an inhibition of the binding of F1 to F0, whereas the proton translocation remained unaffected . Incubation of unstripped everted membrane vesicles with anti-b antibodies resulted in a partial loss of F1, and the remaining membrane-bound ATP-hydrolyzing activity is uncoupled from proton translocation . Similar results were obtained when F(ab')2 or Fab fragments were used . The immunoblot analysis of truncated b' subunits different in length showed that the antigenic determinants are located in the carboxyl-terminal half of the polypeptide chain.

J Biol Chem, 1992 Jun 15, 267(17), 11831 - 8
Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue {2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl}-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T); Balzarini J et al.; {2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl}-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT) . Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer . In contrast with the nonnucleoside derivatives tetrahydroimidazo-{4,5,1-jk}{1,4}- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers . It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase . However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used . TSAO-T does not act as a DNA chain terminator . It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.

Biochem Biophys Res Commun, 1992 Jun 15, 185(2), 683 - 7
Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney; Ross WR et al.; The chaperonin family of proteins, which includes GroEL protein of E . coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial P1 protein . We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA . Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand . After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer . Analysis of column fractions on a 7.5% polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da . Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence . These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5211 - 5
Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells; Chen M et al.; The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C) . A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa) . In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells . This gene was cloned, sequenced, and overexpressed in Escherichia coli . The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP . Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E . coli DNA polymerase III holoenzyme . Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction . Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction . When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone . These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.

J Biol Chem, 1992 Jun 15, 267(17), 12375 - 9
The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides; Hikita C et al.; A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed . The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues . Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency . The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway . It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.

J Biol Chem, 1992 Jun 15, 267(17), 12356 - 63
Purification and characterization of the catalytic domains of the human receptor-linked protein tyrosine phosphatases HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR); Itoh M et al.; Human HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR) are transmembrane receptor-like proteins whose cytoplasmic regions contain either one (HPTP beta) or two (LCA and LAR) domains that are homologous to protein tyrosine phosphatases (PTPases) . Whereas the membrane-proximal domain 1 has enzymatic activity, the membrane-distal domain 2 of both LCA and LAR has no detectable catalytic activity . The cytoplasmic regions of HPTP beta, LCA, and LAR were expressed in Escherichia coli and purified to greater than 90% purity . Modulatory effects of various low molecular weight compounds and homo- and copolymers of amino acids were examined . Several polypeptides that contain a high proportion of tyrosine were strongly inhibitory to these PTPases . To determine a possible role for the LAR domain 2, the properties of recombinant LAR PTPases containing both domains 1 and 2 (LAR-D1D2) or only domain 1 (LAR-D1) were compared . In nearly all aspects examined, LAR-D1 and LAR-D1D2 were indistinguishable . However, polycationic polypeptides strongly stimulated the PTPase activity of LAR-D1D2, but not LAR-D1, using the peptide substrate Raytide . Thus, basic polypeptides seem to indirectly alter the catalytic activity of domain 1 by interacting with domain 2 . This result suggests that domain 2 has a regulatory function.

J Biol Chem, 1992 Jun 15, 267(17), 12244 - 51
Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins . Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins; Maki S et al.; The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD . In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed . Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells . However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain . Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored . Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase . Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction.

Am Surg, 1992 Jun, 58(6), 355 - 7
Cold-induced hypercoagulability in vitro: a trauma connection?
Ferraro FJ Jr, Spillert CR, Swan KG, Lazaro EJ.
Injury severity score and hypothermia can lead to a high level of mortality when combined clinically . In acute trauma, the presence of a coagulopathy is difficult to treat and the aim is prevention . Aliquots of whole blood from healthy human volunteers (n = 9) were added to saline (control) and saline plus endotoxin (activated) . The control and activated groups were divided and subjected to 60 minutes of normothermia (24 degrees C) or hypothermia (0 degrees C) . The samples were returned to 37 degrees C; then the recalcification times were determined using fibrin formation and the viscous drag as the determining factors . The activated hypothermic group showed a decreased recalcification time of 345 (+/- 48.9) seconds compared to 405 (+/- 60.8) for the activated normothermic group (P less than 0.001) . When the normothermic and hypothermic groups were compared without endotoxin added, the differences were not significant . The authors conclude that the effects of endotoxin on clotting time are worsened by hypothermia in vitro and act synergistically to possibly cause the coagulopathy seen in trauma patients.

Am J Respir Cell Mol Biol, 1992 Jun, 6(6), 576 - 82
Suppression of human alveolar macrophage-derived cytokines by amiloride; Rolfe MW et al.; Various human alveolar macrophage (AM)-derived cytokines in the lungs have been shown to be present under conditions of normal homeostasis as well as during the pathogenesis of inflammation . Although extensive investigation has demonstrated the induction of cytokines from AM, relatively little is known regarding endogenous and exogenous regulation of their production . Several pharmacologic agents, including corticosteroids, cyclooxygenase inhibitors, prostaglandins, and methyl-xanthines have been examined for their role in the modulation of mononuclear phagocyte-derived cytokines . In this study, we examine the role of amiloride for the regulation of AM-derived interleukin (IL)-8, tumor necrosis factor (TNF), IL-6, and IL-1 beta . Amiloride in concentrations of 10(-4) to 10(-6) M, concentrations capable of being achieved in the distal airways via nebulization, were shown to inhibit lipopolysaccharide-stimulated, AM-derived IL-8 and TNF in both a time- and dose-dependent fashion . In addition, 5-(N,N-hexamethylene) amiloride hydrochloride, an amiloride analogue with specific sodium channel antiport inhibition, resulted in a similar dose-dependent suppression of lipopolysaccharide-stimulated, AM-derived IL-8 production . Furthermore, the suppressive effect of amiloride appeared to be at the level of mRNA for IL-8, TNF, IL-1 beta, and IL-6, whereas steady-state levels of beta-actin mRNA remained unaltered . These findings would suggest that amiloride has a potentially important modulating influence for the regulation of AM-derived cytokines.

J Cell Biol, 1992 Jun, 117(5), 959 - 73
Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum; Roitelman J et al.; We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway . This domain, which was proposed to span the ER membrane seven times (Liscum, L., J . Finer-Moore, R . M . Stroud, K . L . Luskey, M . S . Brown, and J . L . Goldstein . 1985 . J . Biol . Chem . 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols . The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols . We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER . This indicates that a sequence between peptide G and peptide H spans the membrane of the ER . Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER . These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model . The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276 . Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.

Infect Immun, 1992 Jun, 60(6), 2500 - 5
Functional expression of heterologous fimbrial subunits mediated by the F41, K88, and CS31A determinants of Escherichia coli; Korth MJ et al.; F41, K88, and CS31A are fimbrial adhesins associated with enterotoxigenic Escherichia coli . These adhesins are distinct from one another in the composition of their structural subunits and the adherence characteristics associated with their expression . Despite these differences, extensive homology exists between the genetic determinants mediating the expression of these adhesins, extending throughout the region of each determinant encoding the accessory proteins involved in adhesin biogenesis . This suggests that the regulatory and assembly systems mediating expression of these adhesins may be functionally interchangeable . In the present study we demonstrated that the accessory systems of the F41, K88, and CS31A determinants are able to mediate the functional expression of heterologous fimbrial subunit proteins . Plasmid constructs containing the isolated fimbrial subunit gene of the F41 or CS31A determinant were prepared and introduced into E . coli harboring the F41, K88, and CS31A accessory genes contained on compatible plasmid vectors . The ability of each of the three accessory systems to mediate stable expression of the F41 or CS31A fimbrial subunit peptide was demonstrated by Western blot (immunoblot) analysis . Functional expression of the F41 or CS31A subunit on the bacterial cell surface was demonstrated by the ability of these proteins to confer mannose-resistant hemagglutination of human erythrocytes or in vitro adherence to epithelial cells, respectively . The accessory system of an unrelated adhesin determinant, F1845, did not mediate functional expression of F41 adherence . Taken together, these data indicate that the genetic determinants mediating expression of the F41, K88, and CS31A adhesins are members of a closely related family and suggest that a mechanism exists in the family for the more rapid divergence of genes encoding antigenic and adhesive determinants.

Nucleic Acids Res, 1992 Jun 11, 20(11), 2711 - 6
A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases; Pisani FM et al.; The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes . They were designed on the basis of partial enzyme amino acid sequences . The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa . By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions . By computer-assisted homology search, several sequence similarities among S . solfataricus and family B DNA polymerases were found . In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E . coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified . This result suggests that the proofreading domains of all these enzymes are evolutionarily related.

Nucleic Acids Res, 1992 Jun 11, 20(11), 2627 - 37
How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography; Lim V et al.; Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations . In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA . A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration . In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit . The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit . The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction . These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit . The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation . This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome.

Nature, 1992 Jun 11, 357(6378), 513 - 5
Functional contacts of a transfer RNA synthetase with 2'-hydroxyl groups in the RNA minor groove; Musier-Forsyth K et al.; The functional analysis of determinants on RNA has been largely limited to molecules that contain naturally occurring ribonucleotides, so little is known about the role of 2'-hydroxyl groups in protein-RNA recognition . A single base pair (G3.U70) in the acceptor stem of tRNA(Ala) is the principal element for specific recognition by Escherichia coli alanine-tRNA synthetase . This tRNA synthetase aminoacylates small RNA helices that contain the G3.U70 base pair . Furthermore, removal of the G3 exocyclic 2-amino group that projects into the minor groove eliminates aminoacylation . This 2-amino group is flanked on either side by ribose 2'-hydroxyl groups that line the minor groove . Here we use chemical synthesis to construct 32 helices that make deoxy and O-methyl substitutions of individual and multiple 2'-hydroxyl groups near and beyond the G3.U70 base pair and find that functional 2'-hydroxyl contacts are clustered within a few angstroms of the critical 2-amino group . These contacts are highly specific and make a thermodynamically significant contribution to RNA recognition.

Nucleic Acids Res, 1992 Jun 11, 20(11), 2777 - 84
Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region; Balke V et al.; Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein . To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids . KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis . The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation . This overlaps a known strong C-binding site (at -35 to -54) . RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites . (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2 . RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites . (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed . In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21 . With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C . We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.

Nucleic Acids Res, 1992 Jun 11, 20(11), 2847 - 52
Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation; Sherman JM et al.; The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs . Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2) . Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation . We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase . In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation . Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu) . Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA . The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation . In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E . coli tyrosyl-tRNA synthetase.

Biochim Biophys Acta, 1992 Jun 10, 1135(2), 165 - 70
Binding of endotoxin to macrophages; interactions of spin-labelled saccharide residues; Jackson SK et al.; The molecular mechanisms of endotoxin action are poorly understood . A prerequisite to cellular activation by this agent must be interaction (binding) with the plasma membrane . In this study we have investigated the role of the polysaccharide region of endotoxin (LPS) in binding to macrophages and macrophage-like cell lines . The LPS molecules, from Escherichia coli O111.B4, J5 and the lipid-A, were spin labelled with 2,2,6,6-tetramethylpiperidine-N-oxyl} (Tempo) free radical in their sugar residues, and examined by electron spin resonance spectroscopy . This is the first report of the synthesis of spin-labelled endotoxins . Measurement of the rotational correlation times (Tc) indicated that the saccharide resides do not bind to membrane surface structures and suggests that the binding of LPS to macrophages is mediated by the lipid acyl chains . Anti-sera to LPS from E . coli O111.B4 was effective in binding to the polysaccharide of the same LPS bound to the cell surface.

Biochemistry, 1992 Jun 9, 31(22), 5225 - 31
Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity; Pourcher T et al.; Previous studies utilizing site-directed mutagenesis {Pourcher et al . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 468-472} indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important . The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg . Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane . Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type . Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C . Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature . The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se . Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

Biochemistry, 1992 Jun 9, 31(22), 5215 - 24
Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques; Pelton JG et al.; The 18.1-kDa protein IIIGlc from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars . Phosphorylation of IIIGlc at the N epsilon (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr . NH, 15N, and 13C alpha signal assignments for P-IIIGlc were made through comparison of 15N-1H correlation spectra (HSQC) of uniformly 15N-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-IIIGlc uniformly labeled with both 15N and 13C . Backbone and side-chain 1H and 13C beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-IIIGlc . Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure . The majority of NH, 15N, H alpha, and 13C alpha chemical shifts measured for P-IIIGlc were identical to those obtained for the unphosphorylated protein {Pelton, J . G., Torchia, D . A., Meadow, N . D., Wong, C.-Y., & Roseman, S . (1991) Biochemistry 30, 10043} . Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138 . These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein {Worthylake, D., Meadow, N . D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S.J . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 10382}, and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments . This conclusion is confirmed by comparing NOEs observed in 3D 15N/13C NOESY-HMQC spectra of the two forms of the protein . No NOE differences are seen for residues having the same chemical shifts in IIIGlc and P-IIIGlc . Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in IIIGlc and P-IIIGlc . In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 A, were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1992 Jun 9, 31(22), 5165 - 71
Solution studies on the structure of bent DNA in the cAMP receptor protein-lac DNA complex; Heyduk T et al.; Cyclic AMP receptor protein is involved in the regulation of more than 20 genes . A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein . The induced DNA bending and a structure of the protein-DNA complex were studied by fluorescence energy transfer in 50 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.8 and 20 degrees C . The symmetry of the DNA bend was estimated by measuring the efficiency of transfer between the protein and a label on either the upstream or the downstream end of a lac DNA fragment . The results show that the bend, despite the asymmetry in the DNA sequence, is symmetrical, for the fragments which length ranges from 26 to 40 bp . Using fluorescence energy transfer, the extent of DNA bending was estimated by measuring the end-to-end distance of the DNA fragment which was labeled with a donor-acceptor pair on two opposite ends . Both steady-state and time-resolved measurements showed that in a 26 bp lac DNA fragment complexed with cyclic AMP receptor protein, the end-to-end distance is about 77 A which corresponds to a bending angle of 80 degrees or 100 degrees, depending on the actual contour length between the fluorophores in the free DNA fragment . The results using longer DNA fragments show no measurable amount of energy transfer; thus, it is very unlikely that the DNA completely wraps around the CRP molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jun 9, 31(22), 5158 - 65
Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing . 2 . DNA renaturation reaction; Menge KL et al.; We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA) . In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products . ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation . The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP . Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates . These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates . Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.

Biochemistry, 1992 Jun 9, 31(22), 5093 - 9
Site-directed conversion of a cysteine to aspartate leads to the assembly of a {3Fe-4S} cluster in PsaC of photosystem I . The photoreduction of FA is independent of FB; Zhao J et al.; The terminal electron acceptors FA and FB exist as two {4Fe-4S} clusters located on the 8.9-kDa PsaC protein in photosystem I . We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the {4Fe-4S} clusters were changed to aspartic acid residues . The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp . PCC 6301 in the presence of the PsaD protein . In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact {4Fe-4S} cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a {3Fe-4S} cluster . The {3Fe-4S} cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K . With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered {4Fe-4S} cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand . The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1992 Jun 5, 225(3), 897 - 907
Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions; Despons L et al.; As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast . Efficient aminoacylation of the E . coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence . We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E . coli methionine enzyme . Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation . Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity . The analysis of the L658A mutant shows that Km is mainly affected . This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase . We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663 . Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation . The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced . We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.

J Mol Biol, 1992 Jun 5, 225(3), 609 - 20
Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity; Oertel-Buchheit P et al.; The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding . Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding . In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein . With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84 . The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73 . These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors . In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis . With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor . The most strongly improved variant (LexA EK71, i.e . Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay . As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA . A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor . A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.

J Biol Chem, 1992 Jun 5, 267(16), 11539 - 47
Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway; Hoopes BC et al.; The eukaryotic transcription factor TFIID recognizes and binds a promoter sequence element called the TATA box . We have analyzed the interaction of yeast TFIID with the consensus TATA box sequence of the adenovirus major late promoter . To facilitate this detailed characterization, we developed a method for obtaining quantitative information from a gel retardation (bandshift) assay, allowing measurement of the rate and extent of TFIID-TATA box complex formation . Using this assay and DNase I protection assays, we determined that the association rate constant for TFIID binding to the major late promoter was too low to be consistent with a simple diffusion-limited association, suggesting that the binding proceeds by a multi-step pathway . Furthermore, we found that the slow rate of TFIID binding reported by other research groups was not the consequence of a rate-limiting conformational change, as has been previously suggested . Instead, we observed that the formation of a stable TFIID-TATA box complex was relatively rapid (complete in less than 1 min) at saturating concentrations of TFIID . We have proposed a two-step pathway consistent with the observed kinetics and have considered the possible contributions of each step to the overall rate of TFIID binding . This study lays the groundwork for a systematic characterization of the interaction of TFIID with additional TATA box sequences, including an experimental test of the possibility that different steps in the binding reaction are rate-limiting for different promoters.

J Biol Chem, 1992 Jun 5, 267(16), 11520 - 4
Binding of RepE initiator protein to mini-F DNA origin (ori2) . Enhancing effects of repE mutations and DnaJ heat shock protein; Kawasaki Y et al.; Replication of mini-F plasmid in Escherichia coli requires the plasmid-encoded RepE initiator protein and a number of host factors and is regulated by interaction of RepE with specific sequences near the replication origin, ori2 . We have examined DNA binding properties of several hyperactive mutant RepE proteins with single amino acid substitutions . Plasmids carrying these (repE) mutations, unlike the parental plasmid, can replicate in bacterial hosts lacking the heat shock sigma factor (sigma 32) or deficient in the DnaK, DnaJ, or GrpE heat shock protein . Using gel-retardation assays, the mutant RepE proteins were shown to bind the ori2 repeated sequences with much increased affinities compared to the wild type RepE, whereas they bound to the repE operator with slightly reduced affinities . These results agreed well with the properties of mutant RepE proteins studied in vivo and accounted for the high RepE initiator activities and the high copy numbers of mutant plasmids . In addition, the DnaJ heat shock protein was found to markedly enhance the binding of wild type RepE to ori2 or the operator . DnaK protein with or without ATP failed to show such enhancements . Thus, among the heat shock proteins required for mini-F replication, DnaJ appears to play a major role in RepE binding to ori2 and the operator, perhaps accompanied by RepE activation.

J Biol Chem, 1992 Jun 5, 267(16), 11455 - 61
Rat cystathionine beta-synthase . Gene organization and alternative splicing; Swaroop M et al.; We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene . The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons . These are alternatively spliced, forming four distinct mRNAs (types I through IV) . The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively . Exons 13 and 16 are used alternatively and mutually exclusively . Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV . Exon 16 is present only in type I . Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver . Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III . These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells . Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms . Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E . coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33% . Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.

J Biol Chem, 1992 Jun 5, 267(16), 11176 - 82
The role of negative supercoiling in Hin-mediated site-specific recombination; Lim HM et al.; A series of biochemical assays were developed and performed to monitor the molecular events that occur during the Hin-mediated DNA inversion reaction . These events can be divided into five different stages: 1) binding of proteins (Hin, Fis, and HU) to DNA; 2) pairing of Hin-binding sites; 3) invertasome formation; 4) DNA strand cleavage; 5) strand rotation and religation . A series of topoisomers of the wild type DNA substrate plasmid (ranging from fully relaxed molecules to those with more than the physiological superhelical density (the physiological superhelical density of pKH336 from Escherichia coli DH10B is -0.072 in this study)) was generated, and the role of negative supercoiling in each step of the inversion reaction was investigated . We found differences in the dependence of the formation of paired Hin-binding sites and of the invertasome formation on the superhelical density of the substrate plasmid . Pairing of Hin-binding sites occurs independently from invertasome formation, and a relatively low degree of negative supercoiling is enough to promote maximal pairing . However, efficient invertasome formation requires higher levels of negative supercoiling.

J Biol Chem, 1992 Jun 5, 267(16), 11017 - 22
Insertion and folding of the amino-terminal amphiphilic signal sequences of the mannitol and glucitol permeases of Escherichia coli; Portlock SH et al.; Peptides which correspond to the NH2-terminal 23 or 22 residues of the mannitol and glucitol permeases (enzymes IImtl and IIgut of the bacterial phosphotransferase system; mtl-23 and gut-22) and which are believed to function in envelope targeting were synthesized chemically, and their interactions with lipid model membranes were studied . Both wild-type peptides penetrated phospholipid monolayers up to high surface pressures, and partition constants of 8.0 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively, were derived from the incorporation isotherms of mtl-23 and gut-22 with monolayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine at 32 mN/m or bilayers of the same lipid . The mtl-23 peptide was highly alpha-helical in trifluoroethanol, sodium dodecyl sulfate, lysolecithin, or vesicles of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol, with estimated percentages of alpha-helix ranging between 60 and 85% . The interactions with model membranes of several single site mutants (S3P, D4P, and D4K) of mtl-23 which were defective in properly assembling the mannitol permease in the cytoplasmic membrane of Escherichia coli were also studied . The contents of alpha-helix of these peptides in detergent micelles or phospholipid bilayers were not significantly changed compared with those of the wild type, suggesting that the amphiphilic NH2-terminal membrane-targeting domain could still be formed in these mutants . However, the mutants which contained a proline in positions 3 or 4, i.e . NH2-terminal to the proposed amphiphilic alpha-helix, partitioned into phospholipid monolayers with partition constants that were 2 or 4 times smaller than those of the wild type . Based on these data, a model of the amphiphilic structure of the NH2-terminal domain of the mannitol permease is discussed . This domain may interact physiologically with amphiphilic interfaces of lipids and/or proteins during membrane insertion.

J Biol Chem, 1992 Jun 5, 267(16), 11431 - 8
Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins; Bonner CA et al.; An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex . In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold . As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein . Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event . The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min . We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E . coli DNA polymerases I, II, and III . All three polymerases are extremely inefficient at bypass of the abasic lesion . We find limited bypass by pol I with no change upon addition of accessory proteins . pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB . pol III shows no significant bypass of the abasic site with or without beta,gamma complex.

J Biol Chem, 1992 Jun 5, 267(16), 11606 - 11
Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin; Ehrlich HJ et al.; Plasminogen activator inhibitor 1 (PAI-1), a member of the serpin superfamily of proteins, has been demonstrated previously to interact functionally with the glycosaminoglycan heparin (Ehrlich, H.J., Keijer, J., Preissner, K . T., Klein Gebbink, R., and Pannekoek, H . (1991) Biochemistry 30, 1021-1028) . Heparin specifically enhances the rate of association between PAI-1 and thrombin about 2 orders of magnitude, whereas no effect is detected with other serine proteases (e.g . factor Xa) . For the heparin-dependent serpins antithrombin III and heparin cofactor II, basic amino acid residues in and around the helix D subdomain were proposed to be involved in the binding of glycosaminoglycans . Here we employed site-directed mutagenesis of full-length PAI-1 cDNA to identify the amino acid residues that mediate heparin binding . To that end, 15 single-point mutants of PAI-1, each having individual arginyl, lysyl, or histidyl residues replaced by a neutral (alanyl) residue ("ala-scan"), and one double mutant were constructed, expressed in Escherichia coli, and purified to apparent homogeneity . The purified biologically active proteins were subjected to the following analyses: (i) heparin-dependent inhibition of thrombin; (ii) heparin-dependent formation of sodium dodecyl sulfate-stable complexes with thrombin; and (iii) binding to and elution from heparin-Sepharose . Based on the data presented, we propose that the amino acid residues Lys65, Lys69, Arg76, Lys80, and Lys88 constitute major determinants for heparin binding of PAI-1 . These residues are located in and around the helix D domain and are conserved in the other heparin-dependent thrombin inhibitors, antithrombin III and heparin cofactor II.

J Biol Chem, 1992 Jun 5, 267(16), 11579 - 85
Cauliflower mosaic virus reverse transcriptase . Activation by proteolytic processing and functional alteration by terminal deletion; Takatsuji H et al.; We have previously expressed the cauliflower mosaic virus (CaMV) reverse transcriptase (RTase) gene, the ORFV gene, in yeast in an active form (RTase-Y) . An activity gel analysis revealed that the molecular size of RTase-Y as well as an RTase associated with the CaMV particles (RTase-V) is 60 kDa . This size is about 18 kDa smaller than that of the inactive form previously expressed in Escherichia coli (RTase-E) (78 kDa), which corresponds to the coding capacity estimated for the ORFV gene . To investigate the possible involvement of proteolytic processing in the de novo synthesis of CaMV RTase, we constructed a series of deletions from either terminus or both termini of the ORFV coding sequence and expressed them in E . coli . Among the various truncated RTases, those (denoted delta N) that lack N-terminal peptide fragments 143-185 amino acids long were active on the synthetic RNA template-primer, poly(rC)-oligo(dG) . Those RTases (denoted delta C) lacking C-terminal peptide fragments 50-102 amino acids long and those lacking both termini (denoted delta NC) were also active on this template . However, only the delta N RTases showed enzyme properties indistinguishable from the RTase-Y in that they transcribed natural RNA into DNA and required either Mg2+ or Mn2+ for their activity . The length of the deletion corresponded approximately to the difference of the molecular weights between RTase-Y and RTase-E . These results suggest that CaMV RTase is translated in an inactive precursor form and then converted to an active form by proteolytic processing during de novo synthesis . We have also demonstrated that C-terminal deletions cause a loss of activity on a natural RNA template accompanied by an alteration in metal ion requirement . The inability to incorporate dTTP accounts for the loss of activity on the natural RNA template . However, the affinities for dTTP and the corresponding template, poly(rA)-oligo(dT), were found to be unaltered.

J Biol Chem, 1992 Jun 5, 267(16), 11085 - 91
The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase; Lerner DR et al.; Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin . To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe . The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids . Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin . These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain . The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses . RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems . Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli . This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

J Biol Chem, 1992 Jun 5, 267(16), 10942 - 5
Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25; Jin YJ et al.; Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin . Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity . The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids . Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences . The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12 . The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM) . PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506 . These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin.

J Biol Chem, 1992 Jun 5, 267(16), 11126 - 30
Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli; Gebler JC et al.; The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme . Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined . The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme . The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide . This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G . (1984) Eur . J . Biochem . 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide . A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.

J Biol Chem, 1992 Jun 5, 267(16), 11379 - 85
Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein; Hiasa H et al.; Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro . In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome . When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion . However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region . With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%) . On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate . These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.

J Biol Chem, 1992 Jun 5, 267(16), 11064 - 8
Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli; Pinner E et al.; In Escherichia coli, expulsion of sodium ions is driven by proton flux via at least two distinct Na+/H+ antiporters, NhaA and NhaB . When the nhaA gene is deleted from the chromosome, the cell becomes sensitive to high salinity and alkaline pH (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S . (1989) J . Biol . Chem . 264, 20297-20302) . In the current work we cloned the nhaB gene by complementation of the delta nhaA strain . The gene codes for a membrane protein 504 amino acids long . Hydropathic analysis of the sequence indicates the presence of 12 putative transmembrane helices . NhaB has been specifically labeled with {35S}methionine; it is a membrane protein and displays an apparent M(r) of 47,000, slightly lower than that predicted from its amino acid sequence . Membranes from cells containing multiple dose of nhaB display enhanced Na+/H+ antiporter activity, as measured by the ability of Na+ to collapse a preformed pH gradient or by direct measurement of 22Na+ fluxes . In contrast to NhaA, whose activity increases with pH, NhaB is practically insensitive to pH . Limited homologies with Na+ transporters have been identified.

J Biol Chem, 1992 May 25, 267(15), 10481 - 8
Molecular and immunological characterization of ADP-ribosylarginine hydrolases; Moss J et al.; Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively . Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis . Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent . A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels . Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains . A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes . Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase . A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity . A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA . The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Biochemistry, 1992 Jun 2, 31(21), 5022 - 32
Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway; Meyer E et al.; Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) . The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity . PurE separates from PurK in the first ammonium sulfate fractionation during the purification . No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained . To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity . The activities of PurE, PurK, and PurE.PurK have been investigated . PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate . The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR . No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP . Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi . No dependence on the concentration of HCO3- could be demonstrated . A new energy requirement in the purine biosynthetic pathway has been established.

Biotechniques, 1992 Jun, 12(6), 864 - 9
Expression of a heterodimeric Fab antibody protein in one cloning step; Mullinax RL et al.; A new method is presented for creating antibody expression libraries in Escherichia coli . Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation . This greatly simplifies the construction of antibody expression clones.

Biotechniques, 1992 Jun, 12(6), 804 - 6, 808
High-efficiency cDNA cloning: a comparison of electroporation and in vitro packaging; Gruber CE; The production of complete cDNA libraries from minimal amounts of starting material (i.e., mRNA) is a major challenge for cDNA cloning technology . This paper reports the cDNA cloning efficiencies of electroporation and in vitro packaging . These two methods produce a tremendous number of recombinant clones (greater than or equal to 1 x 10(8) clones/micrograms cDNA) although electroporation generates more clones at nearly all cDNA concentrations used in a ligation reaction.

Parasitology, 1992 Jun, 104 ( Pt 3), 397 - 405
A gene associated with cell division and drug resistance in Giardia duodenalis; Upcroft JA et al.; A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650-800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range . The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock . G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA . Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4 . The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite . In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture . Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

Immunology, 1992 Jun, 76(2), 225 - 8
Induction of IgE antibodies and T-cell reactivity to ovalbumin in rats colonized with Escherichia coli genetically manipulated to produce ovalbumin; Dahlman A et al.; The immune response to ovalbumin (OA) and the bacterial antigens, lipopolysaccharide (LPS) and fimbriae were studied in conventional rats colonized from birth with an Escherichia coli strain producing OA . The colonized rats had developed IgE antibodies against OA, but not against the fimbrial or the LPS antigens from the E . coli at 2 months of age . At this time all rats were primed with OA given intracutaneously in Freund's complete adjuvant . Two weeks later the colonized rats showed a 35% greater delayed-type hypersensitivity (DTH) reaction to OA, measured as ear swelling, than the controls . Thus bacteria carrying antigens resembling potential allergens might aggravate, or participate in the induction of allergic symptoms . In addition such bacteria could be efficient vaccine vectors in protection against parasites . The study illustrates the importance of the mode of antigen presentation for the subsequent immune response.

J Appl Physiol, 1992 Jun, 72(6), 2219 - 24
In vivo effects of Escherichia coli endotoxemia on diaphragmatic microcirculation in rats; Boczkowski J et al.; We investigated the effects of Escherichia coli endotoxin administration on diaphragmatic microcirculation in rats by in vivo videomicroscopy . Rats were allocated into three groups: 1) intravenous inoculation of 10 mg/kg of E . col endotoxin (group E, n = 25), 2) intravenous inoculation of sterile 0.9% NaCl (group C, n = 20), and 3) induction of a controlled hemorrhage by reducing the vascular volume via an arterial catheter (group H, n = 15) . Mean blood pressure (BP) and arteriolar diameters were measured at 15-min intervals and capillary perfusion pattern at 30-min intervals for 1 h . BP decreased similarly in groups E and H, whereas it was maintained in group C . Arterioles were classified as second (A2, n = 46), third (A3, n = 22), and fourth (A4, n = 21) order, according to their relative localization in the network . Basal diameters were the same in the three groups: 38.16, 17.33, and 6.80 microns in group C; 38.17, 17.41, and 7.04 microns in group E; and 37.82, 19.19, and 6.99 microns in group H for A2, A3, and A4, respectively . During the observation period, a significant and similar vasoconstriction of A2 arterioles was observed in groups E and H but not in group C . By contrast, in the three groups, no significant changes in diameter were found for the A3 and A4 arterioles . Capillary perfusion was markedly impaired in group E: at 60 min the percentage of non-perfused capillaries was 40.92 +/- 6.65% in group E compared with 21.17 +/- 5.45% in group C (P less than 0.05) and 18.18 +/- 8.11% in group H (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Int, 1992 Jun, 27(1), 117 - 29
mRNA containing an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1; Balakin AG et al.; The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated . Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied . It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent . It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system . In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.

Arch Dis Child, 1992 Jun, 67(6), 741 - 3; discussion 743-4
The value of proximal small intestinal biopsy in the differential diagnosis of chronic diarrhoea; Thomas AG et al.; The value of proximal intestinal mucosal biopsy was reviewed in 381 children presenting with chronic diarrhoea over an eight year period . An enteropathy was detected in 44% of cases and was more frequently seen in those aged less than 6 months . A diagnosis was established in 91% of cases . The most common diagnosis was the postenteritis syndrome where the presence of an enteropathy indicated those requiring treatment with a cows' milk free diet . Other conditions where a biopsy facilitated diagnosis or treatment included giardiasis, enteropathogenic Escheriichia coli, crytosporidiosis, autoimmune enteropathy, and microvillous atrophy . Coeliac disease was considered in 55% of children and established in 8%, clearly identifying those requiring a gluten free diet . This also emphasises the important role of the biopsy procedure in the exclusion of specific diseases . Proximal small intestinal mucosal biopsy is an essential investigation in children with chronic diarrhoea in whom an enteropathy is suspected.

Vet Microbiol, 1992 Jun 1, 31(2-3), 221 - 33
The pathogenesis of edema disease in pigs . A review; Imberechts H et al.; Edema disease is known to cause important losses in the period shortly after weaning . Although the disease is known for many decades, intensive studies with bacterial lysates of pathogenic E . coli, followed by biotechnological research the last ten years, has led to a better understanding of its pathogenesis . Especially the impact of the toxin is clearly established . Evidence also exists that adhesion factors play a crucial role in the pathogenesis of edema disease.

Mol Microbiol, 1992 Jun, 6(11), 1547 - 53
nov: a new genetic locus that affects the response of Escherichia coli K-12 to novobiocin; Rakonjac J et al.; We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin . The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin . Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E . coli K-12 genetic map . In standard laboratory strains of E . coli K-12 nov exists at least in two allelic forms.

Mol Microbiol, 1992 Jun, 6(11), 1523 - 32
Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli; Sauter M et al.; In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system . The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity . It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA . Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both . None of the mutations affected hydrogenase 1 or 2 activity . A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems . HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

Mol Microbiol, 1992 Jun, 6(11), 1477 - 89
Identification of individual amino acids required for secretion within the haemolysin (HlyA) C-terminal targeting region; Kenny B et al.; The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes . Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, -D and possibly TolC . HlyA is targeted to the medium via the membrane translocation complex, by a novel C-terminal secretion signal . Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20-60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function . In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C-terminus . The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue -46 and which contains at least four individual residues essential for maximal secretion levels . We propose that such residues act co-operatively, forming multiple contact points with the translocator proteins, with the 'best fit' promoting maximal levels of secretion.

J Clin Microbiol, 1992 Jun, 30(6), 1614 - 6
Comparison of a modified adherence assay with existing assay methods for identification of enteroaggregative Escherichia coli; Haider K et al.; Localized, diffuse, and aggregative adherence patterns of Escherichia coli identified with specific DNA probes were compared in cell culture adherence assays by using the Center for Vaccine Development, Baltimore, method, the University of Texas Medical School, Houston (UTH), method, and a modified UTH method . Increasing postwash incubation time from 2 to 4 h in the modified UTH method allowed identification of enteroaggregative E . coli, which was otherwise not identified by the original UTH method.

J Clin Microbiol, 1992 Jun, 30(6), 1585 - 7
Epidemiological analysis of serologically determined rotavirus and enterotoxigenic Escherichia coli infections in Ecuadorian children; Brussow H et al.; The statistical association of rotavirus- and enterotoxigenic Escherichia coli-specific serum antibody with demographic and hygienic factors was tested in Ecuadorian children enrolled in a cross-sectional survey . In 7- to 10-month-old children, enterotoxigenic E . coli-specific antibody was associated (P less than 0.05) with poor drinking water quality, lack of a sewage system, and feeding of supplementary food . In 7- to 14-month-old children, rotavirus-specific antibody was associated only with family size but notably not with hygienic factors.

Gastroenterol Jpn, 1992 Jun, 27(3), 348 - 53
Endotoxin levels in cirrhotic rats with sterile and infected ascites; Sugano S; Endotoxin levels were measured in sterile and bacterially infected ascites in a rat model of phenobarbital and carbon tetrachloride induced cirrhosis was used . An improved chromogenic substrate assay was used to measure endotoxin . All rat ascites specimens were positive for endotoxin . In culture-negative ascites (n = 8), it ranged from 0.05 EU/ml to 0.14 EU/ml (0.08 +/- 0.04 EU/ml, mean +/- SD) (Escherichia coli 0111:B4 endotoxin was used as a reference) . In culture-positive ascites (premortem n = 3, postmortem n = 1), it ranged from 0.78 EU/ml to 1.8 EU/ml (1.29 +/- 0.59 EU/ml, mean +/- SD) . All rats with premortem culture-positive ascites died within two days . This model is useful to study ascites endotoxin levels . In this study, increasing levels of ascites endotoxin correlated with spontaneous bacterial peritonitis and death.

Appl Environ Microbiol, 1992 Jun, 58(6), 2081 - 8
Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry; Tsunekawa H et al.; An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E . coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621 . The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E . coli K-12 derivatives . Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E . coli B-62 . A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+ . A genetically stable Scr+ derivative of E . coli K-12 was obtained by introducing the gene encoding Scr+ onto E . coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation . The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+ . Tryptophan producers E . coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E . coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.

J Med Microbiol, 1992 Jun, 36(6), 398 - 402
Comparison of rapid methods for detection of heat-labile (LT) and heat-stable (ST) enterotoxin in Escherichia coli; Czirok E et al.; Oxoid VET-RPLA, ST-EIA and Pharmacia Phadebact ETEC-LT enterotoxin tests were compared to find a simple but reliable method for detecting enterotoxigenic Escherichia coli (ETEC) in Hungary . In the Oxoid tests, all six reference LT- or ST-producing strains, except one ST-producer, gave positive results . Of 11 reference porcine enterotoxigenic strains, all four LT-producers gave positive reactions for LT but three of 10 ST-producers gave negative reactions for ST . Thirteen of 50 strains from culture collections of H . Steinruck (Germany) were LT+ and nine of 33 were ST+ . When 31 isolates were tested simultaneously with the Oxoid and the Pharmacia LT tests, 12 strains were LT+ by the Oxoid LT test but by the Phadebact LT test only seven of these strains were LT+ and, of the remainder, three gave uncertain results and two gave negative results . Of 69 porcine strains, seven were LT+ and three ST+ . Of 901 human strains isolated in Hungary, 10 were LT+ and one of 24 tested was ST+ . In two cases, ETEC strains were isolated from contacts of travellers returning from Mongolia and Bangladesh . Results of comparative studies with reference strains corresponded well to those of the classical toxin detection tests . The Oxoid test was rapid, sensitive, specific and easy to perform and is recommended for use in screening ETEC isolates.

J Gen Virol, 1992 Jun, 73 ( Pt 6), 1321 - 8
Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes; Kimura N et al.; A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone . Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain . When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.

J Cell Biol, 1992 Jun, 117(6), 1251 - 61
Gas2, a growth arrest-specific protein, is a component of the microfilament network system; Brancolini C et al.; In this report we analyze the protein product of a growth arrest-specific gene, gas2, by means of an affinity-purified antibody raised against the protein produced in bacteria . The regulation of Gas2 biosynthesis reflects the pattern of mRNA expression (Schneider, C., R . King, and L . Philipson . 1988 . Cell . 54:787-793): its relative level is tightly associated with growth arrest . Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism . Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human . We also demonstrate that Gas2 is a component of the microfilament system . It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth-arrested NIH 3T3 cells . The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein . In none of the analyzed oncogene-transformed NIH 3T3 cell lines was Gas2 expression induced under serum starvation.

J Cell Biol, 1992 Jun, 117(6), 1241 - 9
Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC; Doberstein SK et al.; We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail . Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background . The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes . We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA . We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.

FEBS Lett, 1992 Jun 1, 303(2-3), 129 - 35
1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea; Neri D et al.; An E . coli overexpression system for the N-terminal domain of the 434 repressor with residues 1-63 (434 repressor(1-63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine . Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1-63) . In addition, the isotope-labeled tryptic peptide, 44-63, was produced by enzymatic cleavage of the recombinant 434 repressor(1-63), and its NMR spectrum was assigned . Corresponding residues in 434 repressor(1-63) and 434 repressor(44-63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1-69) . These indicate the presence of residual non-random structure in the polypeptide segment 50-60 . The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.

Cancer Genet Cytogenet, 1992 Jun, 60(2), 131 - 4
New chromosomal abnormality . t(1;19;?) in a case of B-chronic lymphocytic leukemia; Montero S et al.; Cytogenetic analysis was performed on peripheral blood cells stimulated with interleukin 6 (IL-6), lipopolysaccharide from Escherichia coli (LPS), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and tetradecanoyl-phorbol-acetate (TPA), in a patient with B-chronic lymphocytic leukemia, showing a t(1;19;?) translocation as the sole abnormality . To our knowledge, this translocation has not been described before in any human neoplasia . In this case, the poor response to therapy (survival time 4 months) suggested that t(1;19;?) could be related to an aggressive course of the disease.

DNA Cell Biol, 1992 Jun, 11(5), 405 - 14
The expression of murine protein disulfide isomerase in Escherichia coli; Haugejorden SM et al.; Protein disulfide isomerase (PDI), a luminal enzyme of the endoplasmic reticulum (ER), is thought to be involved in the process that assures that the correct disulfide bonds form as a newly synthesized protein folds into its appropriate three-dimensional structure (Freeman, 1984) . In recent years, the ER has been shown to have at least two additional, distinct PDI-related luminal proteins (Bennett et al., 1988; Mazzarella et al., 1990) . As a potential first step toward an investigation of the structure and function of PDI and of the PDI-related proteins as well, we have developed a bacterial expression system in Escherichia coli capable of synthesizing significant levels of enzymatically active PDI under the control of the inducible tac promoter . We have observed that the use of this bacterial expression system is complicated by the fact that there is a significant amount of internal initiation of protein synthesis within the PDI coding sequence and the fact that all of the PDI-related expression products are found equally distributed between the cytoplasmic and periplasmic fractions due to a single peptide-independent mechanism . Our studies with this system have demonstrated that at least some truncated PDI molecules containing the carboxy-terminal most active site have significant PDI activity.

Mol Immunol, 1992 Jun, 29(6), 703 - 11
PCR based cloning and sequencing of isogenes encoding the tree pollen major allergen Car b I from Carpinus betulus, hornbeam; Larsen JN et al.; Cloning of the gene encoding the major allergen, Car b I, from Carpinus betulus (hornbeam) pollen was performed using the Polymerase Chain Reaction (PCR) to specifically amplify the gene of interest using single stranded cDNA as template . Specific primers, deduced from the aminoterminal sequence of the purified protein, were tailored to facilitate direct expression of plasmic clones, and the large fraction of positive clones obtained, revealed the presence of isogenic variation . Three clones were characterized in detail by antibody based assays and nucleotide sequencing . The recombinant allergens were shown by crossed immunoelectrophoresis (CIE) to precipitate with monospecific polyclonal rabbit antibodies raised against purified Bet v I, by crossed radioimmunoelectrophoresis (CRIE) to bind tree pollen allergic patient serum IgE, and by immunoblotting to bind murine monoclonal antibodies, raised against purified Car b I from pollen . Car b I is encoded by a 159-triplets open reading frame . The molecular masses (M(r) = 17272, 17355 and 17217 Da, respectively), the amino acid composition, and the aminoterminal sequence of the predicted polypeptides agree well with data obtained by analysis of the protein purified from pollen . The deduced amino acid sequences show pronounced homology (73, 75 and 74% identities respectively) to Bet v I, the major allergen from Betula verrucosa (white birch) pollen . Soluble recombinant Car b I, without a fusion partner, was produced in Escherichia coli with an immunochemical reactivity closely resembling that of the native pollen allergen . The tree pollen major allergens therefore constitute an ideal system for the study of allergenic epitopes.

J Pharmacol Exp Ther, 1992 Jun, 261(3), 964 - 9
Endotoxin-induced activation of cerebral catecholamine and serotonin metabolism: comparison with interleukin-1; Dunn AJ; Administration of either endotoxin (lipopolysaccharide, LPS) or interleukin-1 (IL-1) activates the hypothalamic-pituitary-adrenal axis and cerebral catecholamine systems . Because LPS can stimulate IL-1 production in vivo, it is possible that the effects of LPS are mediated by IL-1 . This hypothesis was evaluated by comparing the neurochemical and corticosterone responses to i.p . LPS and IL-1 . In addition, the possibility that LPS acts by penetrating the brain was examined by comparing the neurochemical responses to i.p . and i.c.v . administration . Intraperitoneal injection of LPS increased mouse brain concentrations of the norepinephrine catabolite, 3-methoxy,4-hydroxyphenylethyleneglycol (MHPG), the dopamine catabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), and the 5-hydroxytryptamine catabolite, 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan in all brain regions examined . By contrast, i.p . IL-1 alpha and IL-1 beta increased cerebral concentrations of MHPG, 5-HIAA and tryptophan, but not DOPAC . The MHPG responses to IL-1 were substantially greater in hypothalamus than in other brain regions, whereas those to LPS were less regionally specific . The minimum effective doses of LPS and IL-1 were around 1 microgram and 10 ng, respectively . After i.p . LPS, plasma concentrations of corticosterone, DOPAC and MHPG peaked around 2 hr, whereas peak concentrations of tryptophan and 5-HIAA occurred around 8 hr . Intracerebroventricular LPS also elevated plasma corticosterone and cerebral concentrations of MHPG and 5-HIAA, but DOPAC was unchanged . LPS was not substantially more potent i.c.v . than i.p.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pharmacol Exp Ther, 1992 Jun, 261(3), 959 - 63
Regional differences in the effects of septic shock on vascular reactivity in the rabbit; Li T et al.; Experiments were performed to investigate the vascular reactivity in both large and resistance artery preparations from an animal model of septic shock . New Zealand White rabbits were injected with a priming dose of Escherichia coli lipopolysaccharide (LPS), 15 micrograms/kg i.v., 18 hr before an i.v . dose of 200 to 2000 micrograms/kg under pentobarbital anesthesia . The second LPS challenge dropped mean blood pressure from 79 +/- 4 to 27 +/- 5 mm Hg in approximately 1 hr . At this time animals were sacrificed with the central ear arteries and kidneys being isolated for alignment in a Krebs-bicarbonate buffer perfusion apparatus to monitor perfusion pressure under constant flow conditions . Individual dose-response curves (DRCs) for norepinephrine (NE) and histamine were performed to assess contractile function . For examining vascular relaxant function, DRCs for methacholine (MC) and nitroprusside (NP) were conducted during a submaximal infusion of NE . The DRCs to NE and histamine were shifted to the right by 2- and 2.7-fold, respectively, in isolated ear artery preparations from LPS-treated vs . vehicle-treated animals . There was no difference in contractile function (using NE) in the two groups of perfused kidneys . The relaxation DRCs to MC were similar in the ear artery preparations from the two treatment groups whereas, in isolated kidneys, the relaxation to MC was significantly attenuated, by an average of 26 +/- 2% at each of six doses, in preparations from LPS-treated animals . The relaxation to NP was similar between the LPS- and vehicle-treated animals in the ear artery and kidney preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Immunol, 1992 Jun, 22(6), 1413 - 9
Cell surface display of rat invariant gamma chain: detection by monoclonal antibodies directed against a C-terminal gamma chain segment; Fisch A et al.; A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant gamma chain (amino acid 142-216) was generated using distinct fusion proteins that contain this gamma segment for immunization and hybridoma screening . Additional fusion protein were prepared carrying discrete regions of the gamma chain . Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis . All mAb established recognize epitopes present on the native gamma chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two-dimensional gel electrophoresis . However, while the majority of the gamma chain-specific mAb precipitated gamma chain-containing polypeptide chain complexes in which immature, sialic acid-deficient and mature, terminally sialylated forms of the gamma chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature gamma forms . Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group . Double-fluorescence staining with the class II-specific fluorescein isothiocyanate-conjugated mAb OX3 and OX6, respectively, as well as a representative gamma chain-specific mAb visualized with phycoerythrin-coupled secondary antibody shows coexpression of class II determinants and the invariant chain at the cell surface.

EMBO J, 1992 Jun, 11(6), 2077 - 85
A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum; Vernon DM et al.; Molecular mechanisms of osmotic stress tolerance were studied in Mesembryanthemum crystallinum (ice plant), a facultative halophyte capable of adjusting to and surviving in highly saline conditions . We screened a subtracted cDNA library enriched for salt stress-induced mRNAs to identify transcripts involved in this plant's adaptation to salinity . One mRNA, Imt1, was found to be up-regulated in leaves and, transiently, in roots . Nuclear run-on assays indicated that this mRNA is transcriptionally regulated . Imt1 encoded a predicted polypeptide of M(r) 40,250 which exhibited sequence similarity to several hydroxymethyl transferases . Expression of the protein in Escherichia coli and subsequent activity assays identified the protein as a novel myoinositol O-methyl transferase which catalyzes the first step in the biosynthesis of the cyclic sugar alcohol pinitol . Pinitol accumulates in salt-stressed M.crystallinum and is abundant in a number of salt- and drought-tolerant plants . The presence of high levels of sugar alcohols correlates with osmotolerance in a diverse range of organisms, including bacteria, fungi and algae, as well as higher plants . The stress-initiated transcriptional induction of IMT1 expression in a facultative halophyte provides strong support for the importance of sugar alcohols in establishing tolerance to osmotic stress in higher plants.

Biochem J, 1992 Jun 1, 284 ( Pt 2), 313 - 9
Cloning and heterologous expression of cDNA encoding class alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic alpha,beta-unsaturated carbonyl compounds; Stenberg G et al.; A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library . The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library . The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained . After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified . Specific activities and kcat./Km values were determined for a number of substrates, including alpha,beta-unsaturated carbonyl compounds . The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation . In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

J Bacteriol, 1992 Jun, 174(12), 4179 - 82
Doublet translocation at GGA is mediated directly by mutant tRNA(2Gly); Pagel FT et al.; Members of the sufS class of -1 frameshift suppressors have alterations of the GGA/G-decoding tRNA(2Gly) . Suppressor-promoted frameshifting at GGA was shown in this study to be directly mediated by the mutant tRNA(2Gly) . We disproved the possibility that, in the presence of the compromised mutant tRNA(2Gly), either wild-type tRNA(1Gly), wild-type tRNA(3Gly), a GGA-reading mutant form of tRNA(3Gly), or any other agent suppresses the frameshift mutation trpE91.

J Bacteriol, 1992 Jun, 174(12), 4166 - 8
Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I; Witkin EM et al.; A polA12 recA718 double mutant of Escherichia coli, in which DNA polymerase I is temperature sensitive, was unable to maintain normal DNA synthesis or to form colonies on rich media at 42 degrees C . Overproduction of DnaE protein, the polymerizing alpha subunit of DNA polymerase III, restored bacterial DNA replication and cell viability, as well as the PolI-dependent replication of the plasmid carrying dnaE.

J Bacteriol, 1992 Jun, 174(12), 3972 - 80
Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli; Iuchi S et al.; In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein . ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side) . In this study, we explored the essential structural features of ArcB by using mutant analysis . The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins . Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB . There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB . The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene . We also showed that the expression of the gene itself is not under respiratory regulation.

J Bacteriol, 1992 Jun, 174(12), 3964 - 71
The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression; Gervais FG et al.; Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated . One of these genes is rcsB, a positive regulator of colanic acid biosynthesis . A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells . Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid . The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB . The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl . Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained . We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.

J Bacteriol, 1992 Jun, 174(12), 3903 - 14
Function of a relaxed-like state following temperature downshifts in Escherichia coli; Jones PG et al.; Temperature downshifts of Escherichia coli throughout its growth range resulted in transient growth inhibition and a cold shock response consisting of transient induction of several proteins, repression of heat shock proteins, and, despite the growth lag, continued synthesis of proteins involved in transcription and translation . The paradoxical synthesis of the latter proteins, which are normally repressed when growth is arrested, was explored further . First, by means of a nutritional downshift, a natural stringent response was induced in wild-type cells immediately prior to a shift from 37 to 10 degrees C . These cells displayed decreased synthesis of transcriptional and translational proteins and decreased induction of cold shock proteins; also, adaptation for growth at 10 degrees C was delayed, even after restoration of the nutrient supplementation . Next, the contribution of guanosine 5'-triphosphate-3'-diphosphate and guanosine 5'-diphosphate-3'-diphosphate, collectively abbreviated (p)ppGpp, to the alteration in cold shock response was studied with the aid of a mutant strain in which overproduction of these nucleotides can be artificially induced . Induction of (p)ppGpp synthesis immediately prior to shifting this strain from 37 to 10 degrees C produced results differing only in a few details from those described above for nutritional downshift of the wild-type strain . Finally, shifting a relA spoT mutant, which cannot synthesize (p)ppGpp, from 24 to 10 degrees C resulted in a greater induction of the cold shock proteins, increased synthesis of transcriptional and translational proteins, decreased synthesis of a major heat shock protein, and faster adaptation to growth than for the wild-type strain . Our results indicate that the previously reported decrease in the (p)ppGpp level following temperature downshift plays a physiological role in the regulation of gene expression and adaptation for growth at low temperature.

J Bacteriol, 1992 Jun, 174(12), 3867 - 73
Identification of the promoter region of the Escherichia coli major cold shock gene, cspA; Tanabe H et al.; The major cold shock protein of Escherichia coli, CS7.4, is produced at a level of 13% of total protein synthesis upon a temperature shift from 37 to 10 degrees C . The transcription of its gene (cspA) was found to be tightly regulated and induced only at low temperature . In addition, the cspA mRNA was extremely unstable at 37 degrees C, so that CS7.4 production was hardly detected when the culture temperature was shifted from 15 degrees C to 37 degrees C . The transcription initiation site (+1) was identified . In vivo footprinting demonstrated that the region from bases -35 to -73 was protected from chemical modification, and gel mobility shift analysis showed that a cold-shocked cell extract contained a factor(s) specifically bound to the fragment containing the sequence between bases -63 and -92 . This factor was synthesized de novo only at low temperature, and its synthesis was inhibited by chloramphenicol . Possible functions of this factor are discussed.

Eur J Biochem, 1992 Jun 1, 206(2), 579 - 85
The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase; Grimm B et al.; Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate . Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases . A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli . The catalytic competence of the resulting enzymes was determined . The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants . Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate . 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form . It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate . The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.

Eur J Biochem, 1992 Jun 1, 206(2), 427 - 35
Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli; Schulze E et al.; Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli . In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively . Chimeric E2p components were constructed by exchanging the three domains between E2p from A . vinelandii and E . coli on gene level . The six chimeric E2p proteins were expressed and purified from E . coli TG2 . All chimeras were catalytically active, 24-subunit E2p proteins . Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A . vinelandii and E . coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex . A . vinelandii E3 interacts only with those chimeras that contain the A . vinelandii binding domain, whereas E . coli E3 interacts with all chimeras . Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3 . Recognition of E1p depends on the origin of both the binding domain and the catalytic domain . E . coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E . coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E . coli E2p . No binding of E . coli E1p was observed when both domains were of A . vinelandii origin . A . vinelandii E1p recognizes E2p from A . vinelandii and E . coli, but strong interaction required that the binding and catalytic domain were of the same origin . Exchange of lipoyl domains had no effect on the binding properties of the E1p component . These observations confirm previous conclusions, based on site-directed mutagenesis of A . vinelandii E2p {Schulze, E., Westphal, A . H., Boumans, H., and de Kok, A . (1991) Eur . J . Biochem . 202, 841-848}, that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E . coli E2p . Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A . vinelandii E2p . Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1992 Jun 1, 206(2), 331 - 6
cDNA cloning of rice lipoxygenase L-2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli; Ohta H et al.; A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings . The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15 degrees C . The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C) . The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence . The molecular mass of the enzyme was calculated to be 96,657 Da based on 865 amino acids . The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50% . A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes . The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km . The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.

Endocrinology, 1992 Jun, 130(6), 3307 - 13
Roles of interleukin-1 alpha and -1 beta in endotoxin-induced suppression of plasma gonadotropin levels in rats; Ebisui O et al.; Using specific antagonists to rat interleukin (IL)-1 alpha and IL-1 beta, the roles of these IL-1s in endotoxin-induced suppression of plasma gonadotropin levels in freely-moving rats were studied . In orchiectomized rats, recombinant rat IL-1 alpha and IL-1 beta administered into the lateral ventricles almost equipotently suppressed plasma LH levels . Twenty five micrograms of bacterial endotoxin or lipopolysaccharide (LPS) administered similarly showed a comparable effect as that of 1 microgram IL-1 alpha or IL-1 beta, and completely lowered plasma LH levels by 60 min after the injection . To examine the roles of endogenous IL-1 alpha and IL-1 beta, anti-rat IL-1 alpha antiserum (anti-IL-1 alpha) and a recombinant human IL-1 receptor antagonist (IL-1ra) were used as specific blockers for IL-1 alpha and IL-1 beta, respectively . Anti-IL-1 alpha (10 microliters) or IL-1ra (10 micrograms) administered intracerebroventricularly (icv) with 25 micrograms LPS, significantly attenuated the LPS-induced effect on plasma LH levels during the first 60 min after LPS infusion, but not during the second 60 min . LPS at a dose of 5 micrograms induced smaller but still significant changes in plasma LH levels compared with 25 micrograms LPS or 1 microgram IL-1 beta . IL-1ra (10 micrograms) completely blocked LH suppression induced by 1 microgram IL-1 beta, but did not completely reverse the changes of LH induced by 5 micrograms LPS . IL-1ra injected iv also significantly attenuated the early suppressive effect of iv administered LPS, but not its late effect on plasma LH levels . However, iv administered IL-1ra had no influence on the effects of icv administered LPS . These data indicate that at least a part of plasma LH suppression caused by icv administered LPS is mediated via IL-1 alpha and IL-1 beta synthesized within the brain, while factor(s) other than IL-1 also participate in the LPS-induced change, particularly during the later period . A similar mechanism may also work peripherally in the case of iv administered LPS-induced plasma LH suppression.

Am Rev Respir Dis, 1992 Jun, 145(6), 1398 - 403
Pulmonary and oxygen transport effects of intravenously administered endotoxin in normal humans; Suffredini AF et al.; Little data are available describing the initial changes in pulmonary function and oxygen transport during human endotoxemia . We studied 26 normal humans after intravenously administered endotoxin (4 ng/kg) . To evaluate alterations in gas exchange, hemodynamic monitoring was performed in nine subjects given endotoxin and six subjects given saline only . Compared with the control subjects, no changes in gas exchange occurred at 3 h, but after volume loading (mean, 2.2 L saline infused from 3 to 5 h) the PaO2 fell (86.1 +/- 2.3 mm Hg, p = 0.042), and the AaPO2 widened (18.5 +/- 1.7 mm Hg, p = 0.005) . Oxygen consumption and delivery both increased significantly at 3 h (219 +/- 17 and 1,030 +/- 43 ml/min.min2) and 5 h (203 +/- 7 and 949 +/- 48 ml/min.min2) (p less than or equal to 0.035), whereas oxygen extraction fell at 3 h (p = 0.041) . Seventeen subjects underwent bronchoalveolar lavage 14 +/- 4 days before and at 1.5 to 3 h (n = 8) or 5 h after (n = 9) the administration of endotoxin . No increase in the total number of cells or percent or absolute number of neutrophils was found at either time point . The rate of clearance of inhaled 99mTc-diethylenetriamine pentacetate aerosol, a measure of alveolar epithelial permeability, increased in subjects scanned before 3 h (n = 8; p less than 0.05), whereas no significant changes occurred in subjects scanned 5 h after endotoxin (n = 5) or in control subjects (n = 6) . Early inflammatory responses after intravenous administration of endotoxin to normal humans results in alterations in gas exchange and lung permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5078 - 82
Structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid R6K; Kelley WL et al.; The plasmid R6K possesses three distinct origins of replication: alpha, beta, and gamma . The replication origin gamma of plasmid R6K performs a dual function: (i) as an origin itself and (ii) as an enhancer element required in cis for the activation at a distance of the other two replication origins alpha and beta . We have dissected the gamma origin/enhancer by site-directed mutagenesis and have reached the following conclusions . The origin function can be specifically inactivated without impairing the enhancer function by insertion and/or deletion mutations near the opposite ends of the origin gamma sequence . One such mutation deleted sequences that included the left DnaA site I . The second mutation involved insertion of linker sequences that resulted in a spatial alteration between the right DnaA site II and the VIIth pi binding iteron (tandemly repeated binding sites) . Other mutations that either partly or completely deleted the A+T-rich sequence adjacent to, but not including, the pi binding iterons also abrogated enhancer and origin function and suggested that pi binding sites were necessary but not sufficient for enhancer activity . Finally, the functional analysis of a set of mutants of the gamma origin/enhancer suggested that a continuous stretch of 300 base pairs is necessary for origin gamma function and that the sequences that included the binding sites for pi, DnaA, and integration host factor proteins are required in the correct stereochemical alignment to impart origin activity.

Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 4864 - 8
Constitutive synthesis of tumor necrosis factor in the thymus; Giroir BP et al.; Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown . If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions . To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence . This construct was inserted into the murine genome, yielding 13 transgenic founders . Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro . Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined . Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium . CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells . CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity . The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.

J Bacteriol, 1992 Jun, 174(11), 3818 - 21
Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase; Shen B et al.; The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity . Expression of the tcmM gene in E . coli results mainly in the TcmM apo-ACP, whereas expression in S . glaucescens yields solely the holo-ACP . The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.

J Bacteriol, 1992 Jun, 174(11), 3715 - 22
DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7(Ts) and dnaK756(Ts); Miyazaki T et al.; The DNA sequence of the dnaK gene of Escherichia coli was analyzed . The nucleotide sequence of the wild-type dnaK gene of E . coli B differed from that of E . coli K-12 in 15 bp, none of which altered the amino acid sequence . Two temperature-sensitive dnaK mutations were examined by cloning and sequence analyses . Results showed that one dnaK mutation, dnaK7(Ts), was a one-base substitution of T for C at nucleotide position 448 in the open reading frame yielding an amber nonsense codon . The other mutation, dnaK756(Ts), consisted of base substitutions (A for G) at three nucleotide positions, 95, 1364, and 1403, in the open reading frame resulting in an aspartic acid codon in place of a glycine codon.

J Bacteriol, 1992 Jun, 174(11), 3667 - 75
Mutational analysis reveals functional similarity between NARX, a nitrate sensor in Escherichia coli K-12, and the methyl-accepting chemotaxis proteins; Collins LA et al.; During anaerobic growth, nitrate induces synthesis of the anaerobic respiratory enzymes formate dehydrogenase-N and nitrate reductase . This induction is mediated by a transcription activator, the narL gene product . The narX gene product may be involved in sensing nitrate and phosphorylating NARL . We isolated narX mutants, designated narX*, that caused nitrate-independent expression of the formate dehydrogenase-N and nitrate reductase structural genes . We used lambda narX specialized transducing phage to genetically analyze these lesions in single copy . Two previously isolated narX* mutations, narX32 and narX71, were also constructed by site-specific mutagenesis . We found that each of these alleles caused nitrate-independent synthesis of formate dehydrogenase-N and nitrate reductase, and each was recessive to narX+ . The narX* mutations lie in a region of similarity with the methyl-accepting chemotaxis protein Tsr . We suggest that the narX* proteins have lost a transmembrane signalling function such that phosphoprotein phosphatase activity is reduced relative to protein kinase activity.

J Bacteriol, 1992 Jun, 174(11), 3651 - 8
Sequence and transcriptional analysis of the Streptomyces glaucescens tcmAR tetracenomycin C resistance and repressor gene loci; Guilfoile PG et al.; Sequence analysis of the tcmA tetracenomycin C resistance gene from Streptomyces glaucescens GLA.O (ETH 22794) identifies one large open reading frame whose deduced product has sequence similarity to the mmr methylenomycin resistance gene from Streptomyces coelicolor, the Streptomyces rimosus tet347 (otrB) tetracycline resistance gene, and the atr1 aminotriazole resistance gene from Saccharomyces cerevisiae . These genes are thought to encode proteins that act as metabolite export pumps powered by transmembrane electrochemical gradients . A divergently transcribed gene, tcmR, is located in the region upstream of tcmA . The deduced product of tcmR resembles the repressor proteins encoded by tetR regulatory genes from Escherichia coli and the actII-orf1 gene from S . coelicolor . Transcriptional analysis of tcmA and tcmR indicates that these genes have back-to-back and overlapping promoter regions.

J Bacteriol, 1992 Jun, 174(11), 3645 - 50
The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family; Sung YC et al.; A regulatory gene, cynR, was found to be located next to the cyn operon but transcribed in the opposite direction . cynR encodes a positive regulatory protein that controls the cyn operon as well as its own synthesis . Positive regulation of the cyn operon requires cyanate and the cynR protein, but the negative autoregulation of the cynR gene appears to be independent of cyanate . The predicted amino acid sequence of the cynR protein derived from the DNA sequence was found to have significant homology to the predicted amino acid sequence of the lysR family of regulatory proteins.

J Bacteriol, 1992 Jun, 174(11), 3558 - 60
High information conservation implies that at least three proteins bind independently to F plasmid incD repeats; Herman ND et al.; The 12 incD repeats in the F plasmid each contain about 60 bits of information, which is three times the amount of conservation that a single protein would need to distinguish the repeats from the rest of the Escherichia coli genome . This is the first reported discovery of a case of threefold excess information, and it implies that at least three proteins bind independently to the repeats . In support of this observation, other workers have shown that three polypeptides bind to this region, but only one, SopB, is known to bind independently of other factors . Identification of the other two proteins should help us to understand the mechanism of plasmid partitioning during cell division.

J Bacteriol, 1992 Jun, 174(11), 3549 - 57
Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3; van Heijenoort Y et al.; The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively . Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification . When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified . Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3 . With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II . It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis . With PBP 3, no polymerization was observed with either substrate . Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed . In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.

J Bacteriol, 1992 Jun, 174(11), 3508 - 13
Transcription in vivo and in vitro of the histone-encoding gene hmfB from the hyperthermophilic archaeon Methanothermus fervidus; Thomm M et al.; Immediately upstream of the hmfB gene, in a DNA fragment cloned from Methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which conforms precisely to the consensus TATA box element proposed for methanogen promoters . By using this duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp downstream from a TATA box, thus providing strong evidence for the functional conservation of this transcriptional signal in two phylogenetically very diverse methanogens . Transcription of the hmfB gene in vivo in M . fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that the genome of M . fervidus contains only one copy of the 73-bp sequence upstream of the hmfB gene . Since the second copy of the 73-bp sequence, presumably generated artifactually during the original hmfB cloning, functioned equally well as a promoter in the M . thermolithotrophicus transcription system, all information needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within this sequence . The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding protein in M . fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures . Cell-free transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1 and 1:1, respectively, whereas the archael histonelike protein HTa from Thermoplasma acidophilum inhibited transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU from Escherichia coli had no detectable effect on transcription.

J Bacteriol, 1992 Jun, 174(11), 3474 - 8
Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins; Sawers G et al.; The pyruvate formate-lyase (pfl) gene of Escherichia coli is transcribed from seven promoters which are coordinately induced 12- to 15-fold by anaerobiosis . The FNR protein plays a major role in the anaerobic control of this system . A mutation in the fnr gene, however, only reduces anaerobic induction fivefold, indicating that FNR is not the only factor involved in the anaerobic activation process (Sawers and Bock, J . Bacteriol . 171:2485-2498, 1989) . The residual anaerobic induction could be shown to be imparted by the transcriptional regulator ArcA; an arcA fnr double mutant was incapable of inducing pfl transcription anaerobically . A mutant strain unable to synthesize the membrane-associated histidine kinase (ArcB) that has been proposed to activate ArcA showed the same phenotype as an arcA mutant strain, indicating that a functional ArcB protein is also required for wild-type, anaerobic pfl transcriptional activation . Nuclease S1 analysis revealed that an arcA mutation abolished anaerobic transcription from promoter 7 and reduced expression from promoter 6 but did not affect transcription from promoters 1 to 5 . On the other hand, an fnr mutation prevented anaerobic expression from promoters 6 and 7 and reduced transcription from promoters 1 to 5 . These data indicate that both ArcA and FNR are essential for anaerobic activation of promoter 7 transcription, which suggests functional interaction between these proteins.

Genes Dev, 1992 Jun, 6(6), 975 - 90
Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif; Nye JA et al.; The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators . Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members . To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays . DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix . Close phosphate and base contacts are restricted to 10 central nucleotides . Contacts map to the major groove in the center of the site . Flanking minor groove interactions also are predicted . To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment . Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain . Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts . These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

Exp Parasitol, 1992 Jun, 74(4), 381 - 9
Plasmodium falciparum: the repetitive MSA-1 surface protein of the RO-71 isolate is recognized by mouse antibody against the nonrepetitive repeat block of RO-33; Olafsson P et al.; We have expressed in Escherichia coli the nonrepetitive repeat zone of the MSA-1 surface protein of the RO-33 isolate of Plasmodium falciparum . The recombinant protein was used to immunize mice and the resulting RO-33 monospecific serum was used to screen our P . falciparum strain collection in order to recover additional alleles lacking tripeptide repeats in block 2 of MSA-1 . Only 1 (RO-71) out of 30 isolates tested reacted strongly with the serum by indirect immunofluorescence assay . Surprisingly, block 2 of the RO-71 MSA-1 allele contains tripeptide repeats resembling those of the K1 isolate of P . falciparum . Additional sequence analysis of the entire DNA coding for the 80-kDa MSA-1-derived surface component did not reveal any amino acid stretches similar to block 2 of RO-33 which could rationalize the immunological cross-reactivity . We eliminated the possibility that the RO-71 culture was contaminated with RO-33 type alleles of MSA-1 by Southern blotting and PCR analysis . The RO-33-specific mouse serum used for the initial selection of RO-71 did not react with the antigen in the denatured state (Western blot) . This and the sequence analysis suggest that the cross-reactive epitope in the MSA-1 protein of RO-71 is conformational . The possibility that a truncated frame-shift protein encoded by mutated MSA-1 mRNA is recognized by the serum is discussed.

Mol Cell Biol, 1992 Jun, 12(6), 2884 - 97
Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element; Kasai Y et al.; The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites . To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension . Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site . Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15 . By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription . The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site . Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites . The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand . Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment . Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts . Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.

J Exp Med, 1992 Jun 1, 175(6), 1717 - 28
Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing; Kaplan G et al.; Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d . With each of these doses and routes, increases in the number of circulating eosinophils were noted . After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d . Reinjection of sites led to enhanced areas of epidermal reaction . GM-CSF prepared from CHO cells was a more potent inducer of this effect . GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes . At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes . These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida . The modified keratinocytes remained MHC class II antigen negative throughout the course of the response . A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells . These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d . During this period the number of epidermal Langerhans cells remained relatively constant . No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route . No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature . 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing . 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls . We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site . There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

Infect Immun, 1992 Jun, 60(6), 2409 - 17
Molecular cloning of epithelial cell invasion determinants from enterotoxigenic Escherichia coli; Elsinghorst EA et al.; Although penetration of the epithelial mucosa has not been identified as a virulence mechanism in enterotoxigenic Escherichia coli (ETEC), we have found that this pathogen is capable of invading human intestinal cell lines . Classical ETEC strain H10407 was most invasive for epithelial cell lines derived from ileocecal and colonic tissues . An ETEC cosmid library was screened for clones that could direct E . coli HB101 to invade cultured human ileocecal epithelial cells (HCT 8 cells) . Three invasive recombinant cosmid clones were isolated . These cosmids could direct HB101 invasion at an efficiency that was equal to or greater than that of the parent ETEC strain . The invasion cosmids also allowed for enhanced HCT 8 cell adherence by HB101 . Electron micrographs of ETEC and recombinant HB101 strains revealed intracellular bacteria contained within endocytic vacuoles . Restriction endonuclease mapping and hybridization analyses showed that the three ETEC clones represent two separate invasion systems present in the parent ETEC strain and that both systems are chromosomally encoded . The parent ETEC strain and one cloned invasion system did not invade HeLa cells . Interestingly, one cloned invasion system was capable of directing HB101 to invade HeLa cells . Invasion of HCT 8 cells by recombinant HB101 strains and the parent ETEC strain was inhibited by cytochalasin D, indicating that the wild-type and both cloned invasion systems trigger an actin polymerization-dependent uptake process . It is not known whether the invasive phenotype of ETEC is relevant for enterotoxigenic disease . However, the parent ETEC strain, as well as recombinant HB101 strains, was capable of transcytosis through polarized HCT 8 monolayers . This transcytosis suggests that ETEC may cross the gut epithelium in vivo and that this invasion may have a previously unrecognized role in the disease process.

Infect Immun, 1992 Jun, 60(6), 2397 - 401
Protection of Aotus monkeys from malaria infection by immunization with recombinant hybrid proteins; Knapp B et al.; On the basis of investigations of the malarial blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum, we chose two Escherichia coli-expressed hybrid proteins containing selected partial sequences of these antigens . Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding P . falciparum polypeptides . In two independent trials with 13 animals, immunization of Aotus monkeys with either of the two hybrid proteins administered in a well-tolerated oil-based formulation protected the animals from an experimental P . falciparum infection.

Infect Immun, 1992 Jun, 60(6), 2252 - 6
Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli; Burnette WN et al.; The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation . The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer . Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin . These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component.

Virology, 1992 Jun, 188(2), 801 - 10
A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope; Engelstad M et al.; Nucleotide sequence analysis of a 42-kb region of the vaccinia virus (strain Western Reserve) genome identified a gene with the potential to encode a 35.1-kDa polypeptide with properties of a membrane glycoprotein (Smith et al., J . Gen . Virol . 72, 1349-1376, 1991) . The 317 amino acid open reading frame (ORF) has similarity with complement control proteins and a secretory vaccinia virus protein (C28K) which interferes with complement function . The predicted B5R gene product differs from the latter protein in that it contains a C-terminal hydrophobic sequence and may be membrane-associated rather than secretory . Transcriptional mapping by Northern blotting and S1 nuclease protection showed that the gene is transcribed both early and late during infection, with the early RNA start site located 60 bp upstream of the late start site that is present at -9 to -5 bp relative to the ORF . Nevertheless, translation of early and late mRNAs are predicted to produce the same polypeptide . A rabbit antiserum was raised to the predicted external hydrophilic domain of B5R expressed in Escherichia coli and used to immunoprecipitate a M(r) 42 K protein from vaccinia-infected cells . This protein was synthesized throughout infection, with a peak from 6 to 7 hr, and its production was inhibited by tunicamycin but not monensin . Western blotting of proteins from purified extracellular enveloped virus (EEV) or intracellular naked virus with anti-B5R serum showed that this M(r) 42 K protein and two higher molecular weight forms (Mr82 and 87 K) were present only in EEV . Anti-B5R serum inhibited comet formation by the IHD-J strain of virus on RK13 cells . B5R is the third vaccinia gene shown to encode an EEV glycoprotein, the others being the virus hemagglutinin gene, and gene SalL4R which encodes a group of lectin-like glycoproteins of M(r) 22-24 K.

Virology, 1992 Jun, 188(2), 486 - 94
A 12,500 MW protein is coded by region E3 of adenovirus; Hawkins LK et al.; There is an open reading frame between ATG291 and TGA612 in the early region E3 transcription unit of adenovirus 2 (Ad2) that could encode a protein of 12,500 MW (12.5K) . To address whether this protein is synthesized, we generated an antiserum against a TrpE-12.5K fusion protein which was expressed in Escherichia coli . This antiserum immunoprecipitated a doublet of about 12.5K apparent MW from {35S}Cys-labeled cells infected with Ad2, Ad5, and various mutants in other E3 genes . Mutants in the 12.5K gene did not produce this protein, and an in-frame deletion mutant showed a protein with a corresponding decrease in size . Cell-free translation of hybridization-purified RNA indicated that 12.5K is coded by E3 mRNA i . mRNA i is relatively scarce, and 12.5K is synthesized in correspondingly small amounts . The 12.5K protein was synthesized at early and late stages of infection in comparable amounts . Pulse-chase experiments indicated that 12.5K has a half-life of about 10 hr . The function of 12.5K is unknown, and the 12.5K gene can be deleted without affecting virus growth in cell culture . However, 12.5K is likely to be important in vivo because the gene is highly conserved in both Ad2 and Ad5 (group C adenoviruses), and also in Ad3 (group B).

Virology, 1992 Jun, 188(2), 459 - 68
Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro; Drelich M et al.; HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure . The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro . Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+ . In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity . The properties of mutant IN proteins were examined in vitro . Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities . The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved . Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro . Mutations of nonconserved residues Gly189 and Thr112 had no effect . Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN . These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.

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