EMBO J, 1993 Jan, 12(1), 279 - 84 Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity; te Heesen S et al.; Asparagine-linked N-glycosylation is an essential protein modification occurring in all eukaryotic cells . The central step is the co-translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn-X-Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum . This reaction is catalyzed by the enzyme N-oligosaccharyl transferase . In yeast, Wbp1p is an essential component of this enzyme . Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation . Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability . Similar to Wbp1p, depletion of Swp1p results in reduced N-oligosaccharyl transferase activity in vivo and in vitro . Wbp1p and Swp1p can be chemically cross-linked, suggesting that both proteins are essential constituents of the N-oligosaccharyl transferase complex. EMBO J, 1993 Jan, 12(1), 265 - 70 SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli; Matsuyama S et al.; The SecD protein is one of the components that has been suggested from genetic studies to be involved in the protein secretion across the cytoplasmic membrane of Escherichia coli . We examined the effect of anti-SecD IgG on protein secretion using spheroplasts . Inhibition of the secretion of OmpA and maltose-binding protein (MBP) by this IgG was observed with concomitant accumulation of their precursor and mature forms in spheroplasts . This effect was specific to anti-SecD IgG . Anti-SecE and anti-SecY IgGs, of which the epitopes are located at the periplasmic domains of SecE and SecY, respectively, did not interfere with the secretion . Time-course experiments investigating the processing of proMBP and the release of MBP from spheroplasts revealed that anti-SecD IgG interfered with the release of the translocated mature MBP . The mature form of MBP thus accumulated was sensitive to trypsin, which was externally added to spheroplasts, whereas MBP released into the medium was resistant to trypsin as the native MBP is . The precursor form of MBP accumulated in spheroplasts was also trypsin resistant . We conclude that SecD is directly involved in protein secretion and important for the release of proteins that have been translocated across the cytoplasmic membrane. EMBO J, 1993 Jan, 12(1), 255 - 63 The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids; Joly JC et al.; To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr) . A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein . Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain . After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase . Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking . The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit. EMBO J, 1993 Jan, 12(1), 243 - 53 Translocation can drive the unfolding of a preprotein domain; Arkowitz RA et al.; Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane . We now find that preprotein unfolding during translocation can be driven by the translocation event itself . At certain stages, translocation and unfolding can occur without exogenous energy input . To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A . At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not . When the ligands are removed from this intermediate, translocation occurs by a two-step process . First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain . This reaction requires neither ATP, delta mu H+ nor the SecA subunit of translocase . Strikingly, this translocation accelerates the net unfolding of the Dhfr domain . In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation . Thus energy derived from translocation can drive the unfolding of a substantial protein domain. Biopolymers, 1993 Jan, 33(1), 147 - 50 Microwave absorption spectroscopy of DNA; Bigio IJ et al.; By utilizing a novel approach to microwave spectrometry, we have measured the absolute absorption spectrum of plasmid DNA (pUC8.c2), in buffered aqueous solution, from 5 to 20 GHz . Our technique does not suffer from the same experimental difficulties that plague other methods . We observe no absorption resonances in this frequency range, but we do see broadband differences, between DNA and pure buffer, that are attributable to changes in the ionic conductivity of the solutions . These results constitute the first verification, by a totally different technique, of the absence of resonances in the microwave absorption spectrum of DNA, and the first data obtained by any technique in the 10-20-GHz band. Ann Rheum Dis, 1993 Jan, 52(1), 32 - 6 Synergism between muramyl dipeptide and lipopolysaccharide in the inhibition of glycosaminoglycan synthesis in cultured rat costal chondrocytes; Ikebe T et al.; The effect of synthetic muramyl dipeptide on glycosaminoglycan synthesis in cultured rat costal chondrocytes was examined . Muramyl dipeptide alone had no effect on the glycosaminoglycan synthesis of rat chondrocytes, whereas Escherichia coli lipopolysaccharide and interleukin 1 alpha inhibited glycosaminoglycan synthesis in a dose dependent manner . Muramyl dipeptide, when added to chondrocyte cultures in the presence of lipopolysaccharide, enhanced the lipopolysaccharide induced inhibition of glycosaminoglycan synthesis in a dose dependent manner . Adjuvant active analogues of muramyl dipeptide, but not adjuvant inactive analogues, also enhanced the lipopolysaccharide induced inhibition of glycosaminoglycan synthesis . In combination with muramyl dipeptide, to inhibit glycosaminoglycan synthesis, lipopolysaccharide could be replaced with the synthetic lipid A, an active principle of lipopolysaccharide . These results show that the muramyl dipeptide portion of bacterial peptidoglycan enhances the susceptibility of rat chondrocytes to the lipid A portion of bacterial lipopolysaccharide, and therefore the interaction between chondrocytes and bacterial cell wall components might be involved in damaging the cartilage in inflammatory joint diseases. Am J Vet Res, 1993 Jan, 54(1), 80 - 5 Cytokine production during endotoxin-induced mastitis in lactating dairy cows; Shuster DE et al.; The role of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha during endotoxin-induced mastitis in cows was characterized . Six cows had 10 micrograms of Escherichia coli lipopolysaccharide infused into 1 mammary gland . Three other cows served as nontreated controls . Within 1.5 to 2.5 hours after infusion, endotoxin caused obvious edema of the mammary gland and increased serum albumin concentration in milk of infused glands 6 times . Milk somatic cell count began to increase 3 to 5 hours after infusion in all treated glands . At 7 hours after infusion, somatic cell counts were increased > 10 times, compared with counts in milk from control cows . Pyrexia of > 1 C developed in only 1 cow, but all treated cows had serum cortisol concentrations > 50 ng/ml in response to endotoxin treatment . High concentrations of IL-1 (10 to 600 U/ml) and IL-6 (2 to 22 U/ml) were detected in milk of infused glands beginning 2.5 to 4 hours after infusion . Endotoxin did not induce detectable amounts of tumor necrosis factor activity in milk or serum . Swelling and mammary gland permeability changes preceded any detectable increase in IL-1 and IL-6 activity, indicating that these clinical signs of inflammation were not mediated by these cytokines . Systemic responses and the leukocytic influx into endotoxin-infused glands developed after or concurrently with initial increases in IL-1 and IL-6 activities in milk . These results suggested that IL-1 and IL-6 may have a role in mammary gland defenses and in the pathophysiologic changes during endotoxin-induced mastitis. Am J Trop Med Hyg, 1993 Jan, 48(1), 26 - 34 Detection of diarrheogenic Escherichia coli in children less than ten years old with and without diarrhea in New Caledonia using seven acetylaminofluorene-labeled DNA probes; Begaud E et al.; We report the use of seven acetylaminofluorene (AAF)-labeled DNA probes in evaluating the incidence of various Escherichia coli pathotypes in New Caledonia among 448 children with acute diarrhea (1,278 E . coli pathotypes studied) and 88 controls (264 E . coli pathotypes studied) in 1990 . Diarrheogenic E . coli were detected using cloned gene probes for heat-labile and heat-stable enterotoxins, Shiga-like cytotoxins (SLTI and SLTII), the cell invasion phenotype (INV), and enteropathogenic-adherence factor (EAF) . Isolates were also studied using bioassays and radioactive DNA probes as reference methods . Enterotoxigenic E . coli (ETEC) were isolated from only 5.36% of the patients; E . coli with localized adherence (LA) to HEp-2 cells was much more common in patients (14.4%) than in controls (3.4%; chi 2 = 7.54, P < 0.01), but most of the E . coli with an LA pattern were members of traditional enteropathogenic E . coli (EPEC) serogroups (chi 2 = 92.95, P < 0.001) . Non-enteropathogenic E . coli with an LA pattern were weakly associated with diarrheal disease (8.9%) . Escherichia coli with a diffuse or an aggregative pattern did not show a significant association with infantile diarrhea . Eight EPEC serogroups were identified and the frequency of positivity for the LA pattern was 70.5%; the EAF was significantly associated with the 0119:K9 serogroup . No enteroinvasive or SLT-producing E . coli were identified . An evaluation of the AAF probes in comparison with 32P-labeled probes and conventional bioassays was made during this epidemiologic survey . The positive and negative predictive values of the ETEC probes were 0.91 and 1, respectively (overall agreement = 99.8%).(ABSTRACT TRUNCATED AT 250 WORDS) Mol Biochem Parasitol, 1993 Jan, 57(1), 89 - 99 A 6-phosphogluconate dehydrogenase gene from Trypanosoma brucei; Barrett MP et al.; A Trypanosoma brucei gene encoding 6-phosphogluconate dehydrogenase (6-PGDH) (EC 1.1.1.44) was identified and cloned by functional complementation of Escherichia coli gnd mutants with genomic trypanosome DNA . The T . brucei gnd gene is present as a single copy . In Northern blot experiments a probe derived from the gene hybridises to 2 transcripts (2.9 kb and 3.1 kb) which are found in both bloodstream and procyclic form organisms; the larger transcript is more abundant in bloodstream form organisms . The derived amino acid sequence of the protein is 479 amino acids in length, with a molecular weight of 52,000 . It is homologous to 6-PGDHs from bacterial and mammalian sources, but diverges significantly from these other enzymes. J Leukoc Biol, 1993 Jan, 53(1), 45 - 52 Selective refractoriness of macrophages to endotoxin-induced production of tumor necrosis factor, elicited by an autocrine mechanism; Fahmi H et al.; Tolerance of macrophages to endotoxin (lipopolysaccharide, LPS) can be induced in vitro by LPS itself . We show here that one of the mechanisms of tolerance to LPS can be mediated via an autocrine process . Continuous exposure to LPS is not required to induce macrophage desensitization . Refractoriness to production of tumor necrosis factor (TNF) in response to LPS can be transferred from tolerant to naive macrophage populations by incubation of the latter with the culture supernatant of the former, in the absence of endotoxin . The active factor present in this macrophage-desensitizing culture supernatant (MD-Sup) is more efficiently removed by incubation with tolerant macrophages than by incubation with naive macrophages . The refractoriness elicited by treatment with MD-Sup is restricted to a decreased TNF response to LPS; interleukin-1 (IL-1) and IL-6 responses are not affected. Invest Radiol, 1993 Jan, 28(1), 39 - 45 Computed tomography and histologic results in the early stages of endotoxin-injured pig lungs as a model for adult respiratory distress syndrome; Muller-Leisse C et al.; RATIONALE AND OBJECTIVES . To determine early radiographic changes in diffuse alveolar injury, the authors correlated computed tomography (CT) and histopathology in pigs with recurrent endotoxinemia . METHODS . Five pigs received recurrent endotoxin over a 17-hour period . Three pigs received physiologic saline and served as controls . Hemodynamic and blood-gas data were analyzed . CT was performed immediately before killing the animals . The lungs were cut into 5-mm-thick slices in the same axis as the CT scans and were investigated by light and electron microscopy . RESULTS . Hemodynamic data, blood-gas analysis, and morphologic changes closely simulated the clinical situation of septic shock in the five pigs that had received endotoxin . Results of histologic examination depicted changes similar to those associated with adult respiratory distress syndrome (ARDS) . CT clearly demonstrated both interstitial, and to a minor degree, intra-alveolar lesions in the endotoxin-injected group, which correlated well with dilated lymph vessels, thickened interstitium, and areas of dystelectasis on histologic examination . Although there was a rather uniform clinical picture, CT and histologic findings showed different degrees of involvement . CONCLUSIONS . CT clearly depicts changes in endotoxin-injured pig lungs in an early clinical state, which are similar to changes associated with ARDS on histologic examination. Diagn Microbiol Infect Dis, 1993 Jan, 16(1), 43 - 51 Identification of species-specific, non-cross-reactive proteins of Borrelia burgdorferi; Sayahtaheri-Altaie S et al.; The low specificity of diagnostic tests for Lyme disease is due to the fact that Borrelia burgdorferi possesses many antigenic proteins that are cross-reactive with other spirochetes and bacteria . The low sensitivity is a result of high (> or = 1:100) dilutions used for patient sera during testing to eliminate non-specific cross-reactivity . The present study was conducted to identify species-specific non-cross-reactive protein(s) of B . burgdorferi that might be used as antigen(s) in serologic tests . Whole-cell sonicates of B . burgdorferi were tested against pooled sera from patients with symptoms, signs, and serologic features diagnostic of Lyme disease (LD), rheumatoid arthritis, infectious mononucleosis, systemic lupus erythematosus, Rocky Mountain spotted fever, secondary syphilis, and from healthy individuals . Different LD pools were also tested against whole-cell sonicates of Treponema pallidum, Treponema phagedenis, Leptospira interrogans, and Escherichia coli . Comparison among patterns obtained by each serum pool revealed that IgM antibodies to species-specific 39-, 23-, and 22-kD proteins and IgG antibodies to 34- and 31-kD proteins were present only in the patients with LD and absent from patients with rheumatoid arthritis, infectious mononucleosis, systemic lupus erythematosus, Rocky Mountain spotted fever, secondary syphilis, and healthy individuals pools . These results suggest that 39-, 23-, and 22-kD proteins may be used in an IgM immunoassay for diagnosis of LD. Cell Immunol, 1993 Jan, 146(1), 96 - 106 T cell lymphokine-induced secretion of cytokines by monocytes from patients with multiple sclerosis; Maimone D et al.; To investigate the function of peripheral blood monocytes in multiple sclerosis (MS), we measured the production of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6), and the procoagulant, tissue factor (TF) in 17 patients with chronic progressive MS and 15 normal controls . Monocyte activity was tested under unstimulated, minimal endotoxin conditions and after culture with various stimuli, including Escherichia coli lipopolysaccharide (LPS), crude supernatant from anti-CD3-activated T cells, recombinant interleukin-2 (rIL-2), and recombinant interferon-gamma (rIFN-gamma) . A higher number of MS patients than controls had circulating monocytes which spontaneously secreted IL-6 or contained detectable cell-associated IL-1 beta . Monocyte responses to LPS were comparable between the two groups; LPS caused production and secretion of all cytokines and TF in every MS patient and control . In contrast, crude T cell supernatants, rIL-2, and rIFN-gamma induced IL-1 beta release in a higher number of MS monocytes than that in controls, whereas the production and secretion of the other cytokines and TF activity were similar between the groups . We conclude that some MS patients have "primed" circulating monocytes, as shown by excessive spontaneous IL-6 release and intracellular IL-1 beta synthesis . Unstimulated MS monocytes, however, are not different from controls with respect to spontaneous secretion of small amounts of IL-1 beta and TNF alpha and expression of cell surface TF . Excessive IL-1 beta secretion by MS monocytes after stimulation with T-cell-derived lymphokines suggests dysregulation of T cell-monocyte interactions which may be most relevant in the central nervous system plaques where activated T cells are found. Plant Mol Biol, 1993 Jan, 21(2), 213 - 24 Structure and evolution of a highly repetitive DNA sequence from Brassica napus; Xia X et al.; A Hind III family of highly repetitive DNA sequences, canrep (canola repeat), was cloned from the nuclear DNA of canola (Brassica napus cv . Westar) . The basic units of this family of repeats consists of 176 bp and are arranged in clusters of tandem direct repeats . Each canrep repeat is composed of three related subrepeats of ca . 60 bp . Each subrepeat contains two inverted repeats of about 23 bp and another unrelated sequence of about 12 bp . Based on the internal structure, a possible scheme for the evolution of canrep is proposed . At least two subfamilies of the canrep sequences are present in the genome, as revealed by sequence analyses . In situ hybridization showed that canrep sequences are mainly clustered at centromeric regions of chromosomes . Northern hybridizations indicate that there are no transcripts related to canrep in the total RNAs extracted from plant seedlings. Plant Mol Biol, 1993 Jan, 21(1), 99 - 108 Large unidentified open reading frame in plastid DNA (ORF2280) is expressed in chloroplasts; Glick RE et al.; The chloroplast DNA encodes genes for components of photosynthesis and the transcription-translation machinery; a number of unidentified open reading frames (ORFs) are also present . To determine whether a large ORF in the inverted repeat of chloroplast DNA of tobacco (ORF2280) encodes a chloroplast protein, a conserved region of the ORF was expressed in Escherichia coli . An antibody against the ORF protein was prepared using the purified fusion protein as an antigen . When incubated with proteins from the soluble fraction of tobacco, spinach and Oenothera chloroplasts, the antiserum detects relatively labile polypeptides, which have apparent molecular weights of 170 to 180 kDa . The ORF in tobacco and spinach is large enough to encode a protein of 240-250 kDa, thus it is possible that post-transcriptional or post-translational processing reduces the size of the expression product . Analysis of Oenothera chloroplasts representing four different plastome types revealed endonuclease restriction fragment length polymorphisms in chloroplast DNA indicative of insertion/deletion events in a region of the chloroplast DNA that shared significant sequence similarity with ORF2280 . The ORF2280 antiserum was used to demonstrate that there are qualitative differences in the ORF proteins from different Oenothera plastome types. Plant Mol Biol, 1993 Jan, 21(1), 185 - 9 A beta-ketoacyl-acyl carrier protein synthase III gene (fabH) is encoded on the chloroplast genome of the red alga Porphyra umbilicalis; Reith M; DNA sequencing of a region of the chloroplast genome of the red alga Porphyra umbilicalis revealed an open reading frame of 326 amino acids . Databank searches indicated that this ORF is 34% identical to an E . coli gene (fabH) encoding beta-ketoacyl-carrier protein synthase III . In addition, a leucine tRNA gene (trnL(GAG)) was detected just downstream . Neither of these genes are encoded on the chloroplast genomes of land plants. Biochem J, 1993 Jan 1, 289 ( Pt 1), 81 - 5 Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex; Quinn J et al.; The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue . A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli . Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species . The apo-domain can be lipoylated in vitro with partially purified E . coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase) . Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains. Arch Biochem Biophys, 1993 Jan, 300(1), 501 - 9 Studies on fibronectin and its domains . I . Novel recombinant cell-binding domain of fibronectin--a modulator of human platelet functions; Vogel T et al.; The DNA sequences encoding for two proteins of the cell-binding domain (CBD) of human fibronectin (FN), namely a 33-kDa protein (aa 1329-1722) and a 40-kDa (aa 1380-1851) protein, were cloned and expressed in Escherichia coli . The interactions of the resulting rCBD proteins, refolded and purified to homogeneity, with human platelets were studied in comparison with those of the pentapeptide GRGDS . The binding of both the 33-kDa and the 40-kDa proteins to washed platelets appeared to be dependent upon platelet activation . In the case of the 33-kDa protein, binding to stimulated platelets was shown to be saturable, with Kd = 2 microM (thrombin as agonist) . Moreover, both the 33-kDa and the 40-kDa proteins inhibited fibrinogen binding (at 0.1 microM) to ADP- or thrombin-stimulated platelets with IC50 values in the same concentration range . Binding seemed therefore to occur mainly at the GPIIb/IIIa receptor, and accordingly monoclonal antibodies against this receptor prevented up to 85% of the binding of the 33-kDa protein to platelets . With most stimuli the 33-kDa and the 40-kDa proteins inhibited platelet aggregation at concentrations 15- to 25-fold lower than those required by GRGDS and, in the case of the 33-kDa protein, this was shown to occur in either platelet-rich plasma, washed platelets, or whole blood . The 33-kDa protein also inhibited platelet aggregation and thromboxane A2 (TXA2) generation on the subendothelial extracellular matrix, whereas the GRGDS peptide inhibited only matrix-induced platelet aggregation, but not TXA2 formation . Furthermore, the 33-kDa protein, which is derived from the human FN CBD, seemed to be highly selective, since it inhibited the aggregation of platelets from primates only, and not from other animals tested . Finally, the 33-kDa protein did not promote fibroblast cell attachment, as was observed for both whole FN and the 40-kDa protein, thus displaying a selectivity toward platelets . In conclusion, the unique properties of the 33-kDa protein, and, in particular, its special affinity directed only toward activated primate platelets, seem to hold a promising potential for the further development of an antithrombotic agent. Arch Biochem Biophys, 1993 Jan, 300(1), 451 - 7 Structural and functional studies on the interaction of sodium dodecyl sulfate with beta-galactosidase; Muga A et al.; The effect of sodium dodecyl sulfate (SDS) on enzyme activity, electrophoretic behavior, and conformation of Escherichia coli beta-galactosidase is presented . Fourier-transform infrared spectroscopy (FT-IR), previously used to study the structure of native beta-galactosidase has been applied to examine the detergent effects on the enzyme . At 20 degrees C, the presence of 1% SDS does not cause appreciable changes in the secondary structure, and enzyme activity is preserved; however, 10% SDS produces complete enzyme inactivation and FT-IR spectroscopy indicates a concomitant change in conformation . Thermal denaturation of beta-galactosidase starts at approximately 53 degrees C in the absence and at approximately 46 degrees C in the presence of 1% SDS, indicating tertiary structure changes; also, a good correlation between structural (FT-IR) and functional (Arrhenius plots) data is observed . The secondary structure of thermally denatured beta-galactosidase contains mainly extended structures, and intermolecular interactions produce protein aggregation . In the presence of 10% SDS, however, the hydrophobic segments of the protein are stabilized by SDS into helical structures without protein aggregation . At 30 degrees C, in the presence of 1% SDS, two protein bands are resolved by gel electrophoresis, only one of them being active . A model for SDS-galactosidase interaction is proposed, according to which, at low surfactant concentrations, SDS molecules bind the outer surface of the protein, without affecting the protein core . Higher detergent concentrations produce a larger conformational change involving enzyme inactivation and increased accessibility of the solvent to the protein core . Increasing temperature in the presence of 10% SDS leads to a facilitated access of surfactant molecules to the inner protein regions and to an increase of the beta-galactosidase alpha-helical content. Arch Biochem Biophys, 1993 Jan, 300(1), 416 - 22 Overproduction of soluble trichodiene synthase from Fusarium sporotrichioides in Escherichia coli; Cane DE et al.; Trichodiene synthase is a sesquiterpene cyclase isolated from various fungal species which catalyzes the cyclization of farnesyl diphosphate (FPP) to trichodiene . The trichodiene synthase gene (Tox5) of Fusarium sporotrichioides has previously been cloned and expressed as 0.05-0.1% of total cell protein in Escherichia coli . We have used polymerase chain reaction to amplify the trichodiene coding sequence carried on the plasmid pTS56-1 . The resulting DNA, carrying a BamHI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon as well as a HindIII site adjacent to the translational stop codon, was inserted into the corresponding sites of the expression vector pLM1 . The latter vector carried the promoter and translational leader sequence from T7 gene 10 and the E . coli rmBT1T2 tandem transcription terminator . This construct was cloned into E . coli BL21 (DE3) . The resulting transformants, when induced with isopropyl beta-D-thiogalactoside, produced trichodiene synthase as 20-30% of total soluble protein . The recombinant synthase, which could be purified five-fold to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and gel filtration on Superose 12, was identical to native protein in steady-state kinetic parameters and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the expected MENFP N-terminal sequence. Arch Biochem Biophys, 1993 Jan, 300(1), 193 - 200 Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase; Chan CL et al.; O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis, and carcinogenesis . Recombinant human MGMT was isolated from an Escherichia coli high performance expression system and purified to homogeneity . The kinetic and DNA-binding properties of the recombinant human MGMT were studied . The purified human MGMT reacted stoichiometrically with methylated DNA under second-order rate kinetics . The rate constant with normal methylated DNA was 1 x 10(9) M-1 min-1 at 37 degrees C . The binding to DNA was the rate determining step in the repair process . Approximately eight base pairs of the DNA substrate were covered by the human MGMT protein . The affinity constant for interaction of DNA to MGMT was approximately 4.7 x 10(5) M-1 . The binding to methylated DNA was also examined; the binding affinity to methylated DNA was two times higher than that to unmodified DNA . The interaction with DNA induced a conformational change in the human MGMT protein as monitored by circular dichroism and fluorescence analysis . A similar conformational change was induced by both methylated and unmodified DNA. Arch Biochem Biophys, 1993 Jan, 300(1), 169 - 77 Porphobilinogen synthase from Escherichia coli is a Zn(II) metalloenzyme stimulated by Mg(II); Mitchell LW et al.; Porphobilinogen synthase (PBGS) is essential to all life forms; in mammals it is definitively established that Zn(II) is required for activity . The literature regarding the metal requirement for PBGS in other animals, plants, and bacteria neither establishes nor disproves a Zn(II) requirement . We have characterized Escherichia coli PBGS and found it to be remarkably similar to bovine PBGS . The similarities include a requirement for Zn(II), inhibition by 1,10-phenanthroline, an exceptional thermal stability, a requirement for free sulfhydryl(s) as shown by sensitivity to modification by methyl methanethiosulfonate, and the presence of tightly bound product on freshly isolated enzyme . Proton-induced X-ray emission analysis shows E . coli PBGS to contain a stoichiometric amount of Zn and no other metals . The most striking similarity between E . coli and bovine PBGS is the 13C NMR spectrum of enzyme-bound {3,5-13C}PBG; the chemical shifts of bound product are identical for both bovine and E . coli PBGS . Minor differences between E . coli PBGS and its mammalian counterpart include Km (ALA) = 1.9 mM, a pH optimum of 7.5-8, and its molar absorbtion coefficient expressed as A(0.1%)280 is 0.588 . We conclude from these data that E . coli PBGS is a Zn(II)-metalloenzyme and that Zn(II) is required for catalytic activity, and propose that the mammalian and bacterial PBGS function by similar mechanisms . There is one significant difference between E . coli and mammalian PBGS . For E . coli PBGS, Mg(II) causes a twofold stimulation of the Zn(II)-induced E . coli PBGS activity; this effect is not seen for bovine PBGS . The stimulation of activity by Mg(II) mimics the effect of Mg(II) on plant PBGS, although E . coli PBGS does not contain the putative Mg(II) binding site recently revealed by Boese et al . {Q . F . Boese, A . J . Spano, T . Li, and M . P . Timko (1991) J . Biol . Chem . 266, 17060-17066} . This work lays the foundation for identification of functional amino acids based on the sequence similarities between PBGS from bacterial, plant, and mammalian sources. J Gen Virol, 1993 Jan, 74 ( Pt 1), 47 - 53 Viable double vaccinia virus recombinants with the non-inducible phage T7 expression system; Kriajevska MV et al.; Double vaccinia virus recombinants expressing both the T7 RNA polymerase gene, controlled by a weak early poxvirus PF promoter, and the Escherichia coli beta-galactosidase gene, controlled by the phage T7 promoter, have been obtained . The viability of the double recombinants depended on the T7 RNA polymerase expression level . If the T7 RNA polymerase gene was inserted into a recombinant already containing the beta-galactosidase gene, the efficiency of formation of the double recombinants was significantly higher compared to that for the reverse insertion order . The negative effect of the phage T7 terminator on beta-galactosidase expression in cells infected with the recombinant viruses has been shown . The dynamics and levels of beta-galactosidase formation by different vaccinia virus recombinants have been studied. J Clin Invest, 1993 Jan, 91(1), 225 - 34 Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer; Mastrangeli A et al.; A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium . This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated . Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes . To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase {beta-gal}) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats . In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium . The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals . Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis. Genes Dev, 1993 Jan, 7(1), 161 - 72 Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription; Li J et al.; NusG is a transcriptional elongation factor in Escherichia coli that aids transcriptional antitermination by the phage lambda N protein . By using NusG affinity chromatography, we found that NusG binds directly and selectively to termination factor rho . NusG was shown previously to be needed for termination by rho in vivo, and we show here that NusG increases the efficiency of termination by rho at promoter-proximal sites in vitro . The rho026 mutation makes termination by rho less dependent on NusG . It also makes antitermination by N at rho-dependent terminators and the binding of rho to NusG temperature sensitive . Therefore, the interaction of NusG with rho is important both for rho-dependent termination and for antitermination by N at rho-dependent terminators. Genes Dev, 1993 Jan, 7(1), 149 - 60 Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli; Makino K et al.; Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major sigma-factor sigma 70 . To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes . We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties . The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of sigma 70 . Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most . The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters . Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes . These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB. FASEB J, 1993 Jan, 7(1), 168 - 72 Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli; Jakubowski H; Previous in vitro work has shown that Escherichia coli methionyl-tRNA synthetase has a limited ability to discriminate against cognate methionine in the editing site designed for noncognate homocysteine . As a result, a small fraction of the correct product Met-tRNA is deacylated with the formation of a cyclic sulfonium compound, S-methyl-homocysteine thiolactone . This is exploited here to estimate energy costs associated with the destruction of a correct product by methionyl-tRNA synthetase in bacterial cells . In vivo measurements of S-methyl-homocysteine thiolactone indicate that in Escherichia coli 3.3 molecules of Met-tRNA are destroyed by deacylation per 10,000 molecules of Met-tRNA successfully transferring methionine to protein . This number of destroyed molecules of a correct product, Met-tRNA, is 30 times lower than the number of destroyed molecules of an incorrect product, homocysteinyl adenylate . Thus, most of the energy cost of proofreading in vivo is due to editing of the noncognate amino acid. FASEB J, 1993 Jan, 7(1), 143 - 8 Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo; Reuven NB et al.; Our knowledge of the 3' processing of tRNA precursors is severely limited . Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated . Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo . Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness . Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells . These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes . These studies contribute to the identification of all the enzymes necessary for defining the complete processing pathway for E . coli tRNA precursors. Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 168 - 72 The role of zinc fingers in transcriptional activation by transcription factor IIIA; Del Rio S et al.; We have described elsewhere a number of the properties of a set of mutant forms of Xenopus transcription factor IIIA (TFIIIA) containing single amino acid substitutions that result in the structural disruption of individual zinc finger domains . These "broken finger" proteins have now been analyzed with respect to their ability to support transcription of 5S rRNA genes in vitro . Disruption of any one of the first six zinc fingers of TFIIIA has no discernible effect on the activity of the protein in supporting 5S rRNA synthesis in standard in vitro transcription assays, despite the fact that some of these mutant proteins exhibit large decreases in their binding affinity for 5S rRNA genes in binary complexes . These results indicate that the activity of TFIIIA as a transcription factor can be largely independent of its equilibrium binding constant for the 5S rRNA gene in the absence of other components of the RNA polymerase III transcriptional apparatus . In fact, this finding is consistent with the known pathway and kinetics of assembly of 5S rRNA transcription complexes . In contrast to the results obtained with finger 1-6 mutants, analogous mutations in zinc fingers 7-9 of TFIIIA result in moderate to complete loss of transcriptional activity . We interpret these results to mean that the three C-terminal zinc fingers of TFIIIA are not only involved in binding to the internal control region of 5S rRNA genes but are also required, either directly or indirectly, for higher-order interactions that are important in transcription complex assembly, stability, or activity. J Bacteriol, 1993 Jan, 175(2), 568 - 71 Characterization of the gcd gene from Escherichia coli K-12 W3110 and regulation of its expression; Yamada M et al.; DNA sequence and expressional analyses of the gcd gene of Escherichia coli K-12 W3110 revealed that two promoters that were detected were regulated negatively by cyclic AMP and positively by oxygen . Sequence conservation of the gcd gene between E . coli K-12 W3110 and PPA42 suggests that glucose dehydrogenase is required for the E . coli cells, even though it ordinarily exists as an apoprotein. J Bacteriol, 1993 Jan, 175(2), 565 - 7 Membrane topology model of Escherichia coli alpha-ketoglutarate permease by phoA fusion analysis; Seol W et al.; Escherichia coli alpha-ketoglutarate permease (KgtP) is a 432-amino-acid protein that symports alpha-ketoglutarate and protons . KgtP was predicted to contain 12 membrane-spanning domains on the basis of a calculated hydropathy profile . The membrane topology model of KgtP was analyzed by using kgtP-phoA gene fusions and measuring alkaline phosphatase activities in cells expressing the chimeric proteins . Comparisons of the phosphatase activity levels and the locations of the KgtP-PhoA junctions are consistent with the predicted membrane topology model of KgtP. J Bacteriol, 1993 Jan, 175(2), 561 - 4 Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174; Kow YW et al.; In order to examine the possible role of Escherichia coli DNA polymerase II in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded phi X174 transfecting DNA containing individual lesions . No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase II was observed . Furthermore, DNA polymerase II did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint . Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II. J Bacteriol, 1993 Jan, 175(2), 553 - 6 Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions; Boyd D et al.; An approach to analyzing the topology of membrane proteins with alkaline phosphatase fusions is described . Precise fusions were constructed by using polymerase chain reaction at the C terminus of each hydrophilic region of the membrane protein . The disruption of topogenic signals is thereby minimized, and predictable anomalous results are avoided . The Escherichia coli MalG protein has been analyzed. J Bacteriol, 1993 Jan, 175(2), 428 - 37 Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli; Yang YL et al.; We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid . These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold . The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids . Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s) . All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I . The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I . Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid . The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme . Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed. J Bacteriol, 1993 Jan, 175(2), 367 - 76 Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus; Van Way SM et al.; The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, is near the top of the flagellar-gene regulatory hierarchy, and it is one of the earliest transcription units to be expressed in the cell cycle . In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription . The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo . These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A . Dingwall, A . Zhuang, K . Quon, and L . Shapiro, J . Bacteriol . 174:1760-1768, 1992) . We propose that this 18-bp sequence contains all or part of the fliF promoter . We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D . A . Mullin and A . Newton, J . Bacteriol . 171:3218-3227, 1989) . Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation . A 2-bp deletion at -12 and -13 in ftr4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly . We suggest that the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle. J Bacteriol, 1993 Jan, 175(2), 351 - 7 Complementation between nucleotide binding domains in an anion-translocating ATPase; Kaur P et al.; The catalytic component of the oxyanion-translocating ATPase of the plasmid-encoded ars operon of Escherichia coli is a homodimer of the ArsA protein . This enzyme is an oxyanion-stimulated ATPase with two consensus nucleotide binding sequences in each subunit, one in the N-terminal (A1) half and one in the C-terminal (A2) half of the ArsA protein . The two halves of both the arsA gene and the ArsA protein exhibit similar nucleotide and amino acid sequences, respectively . The two halves of the arsA gene were subcloned into compatible plasmids . Neither alone was sufficient to confer resistance, but cells in which the arsA1 and arsA2 half genes were coexpressed were resistant to arsenicals . Genetic complementation was also observed in cells bearing plasmids with point mutations in the two halves of the arsA gene and between cells with plasmids carrying combinations of the arsA1 or arsA2 subclones and point mutations . In every case, complementation was observed only when one plasmid contained a wild-type arsA1 sequence and the other contained a wild-type arsA2 sequence . These results demonstrate that both sites are required for resistance but that the two nucleotide binding domains need not reside in a single polypeptide . We propose a model in which the ArsA dimer has two catalytic units, each composed of an A1 domain from one monomer and an A2 domain from the other monomer. J Bacteriol, 1993 Jan, 175(2), 341 - 50 Characterization of DNA helicase II from a uvrD252 mutant of Escherichia coli; Washburn BK et al.; The loss of DNA helicase II (UvrD) in Escherichia coli results in sensitivity to UV light and increased levels of spontaneous mutagenesis . While the effects of various uvrD alleles have been analyzed in vivo, the proteins produced by these alleles have not been examined in any detail . We have cloned one of these alleles, uvrD252, and determined the site of the mutation conferring the phenotype . In addition, the protein it encodes has been purified to homogeneity and characterized in vitro . The mutation responsible for the phenotype was identified as a glycine-to-aspartic-acid change in the putative ATP-binding domain . In comparison to wild-type DNA helicase II, the UvrD252 enzyme exhibited reduced levels of ATPase activity and a large increase in the Km for ATP . The ability of UvrD252 to unwind DNA containing single-stranded regions, as well as DNA containing only nicks, was reduced in comparison to that of the wild-type enzyme . Possible interpretations of these results in relation to the phenotypes of the uvrD252 mutant are discussed . This represents the first detailed analysis of the biochemical properties of a mutant DNA helicase II protein. Eur J Immunol, 1993 Jan, 23(1), 217 - 23 Leishmania mexicana promastigotes induce cytotoxic T lymphocytes in vivo that do not recognize infected macrophages; Lopez JA et al.; The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules . We used E . coli beta-galactosidase as a model antigen which can be expressed in high levels in L . mexicana promastigotes (L . mexicana-gal) . Infection of BALB/c mice with L . mexicana-gal induces beta-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a beta-galactosidase-expressing mastocytoma line as an antigen-presenting cell . These CTL recognize epitopes of beta-galactosidase in the context of H-2Kd; however, they do not recognize L . mexicana-gal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal . It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules . The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming. Environ Mol Mutagen, 1993, 21(1), 2 - 7; discussion 46-57 Japanese guidelines for mutagenicity testing . Ministry of Agriculture, Forestry and Fisheries; Ministry of Health and Welfare; Ministry of Labor; Sofuni T; Several Japanese agencies are required to perform mutagenicity tests according to regulatory guidelines . Although each agency's guidelines address a specific purpose, the experimental principles behind them are similar, and general methodological recommendations have been issued by the Ministry of Agriculture, Forestry, and Fisheries {1985}; Ministry of Health and Welfare {1990}; Ministry of Labor {1991}; and Ministry of Health and Welfare {1992} . Four major guidelines for mutagenicity testing in Japan and some amendments are briefly introduced . In addition, several procedures in Japanese guidelines that differ from those of other countries or organizations are discussed. Arch Virol, 1993, 128(1-2), 15 - 27 Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene; Ohshima K et al.; Complete nucleotide sequences of cytoplasmic inclusion (CI) genes of two strains of potato virus Y (PVY) were determined from six polymerase chain reaction (PCR)-amplified cDNA clones . The size of the CI genes of both ordinary (PVY-O) and necrotic strains (PVY-T13) was 1902 nucleotides, with a sequence homology of 83.4% . Comparison of the predicted amino acid sequences showed more than 90% homology . When these were compared with those of other potyviruses, the homology ranged from 53 to 61% . cDNAs of all or a part of the PVY-O CI gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and cloned into an Escherichia coli expression vector, pKK 223-3 . Complete and truncated PVY-O CI proteins were successfully produced in E . coli as judged by reactivities with PVY-O CI protein-specific antiserum . To our knowledge, this is the first report on expression of PVY CI proteins in E . coli. Science, 1993 Jan 1, 259(5091), 84 - 7 Regulation of the human hsp70 promoter by p53; Agoff SN et al.; The tumor suppressor p53 is a nuclear phosphoprotein with characteristics of a transcription factor . It displays sequence-specific DNA binding, contains a potent transactivation domain, and has been implicated as both a transcriptional activator and a repressor . Transcription of the human hsp70 gene is stimulated by adenovirus E1a protein . This E1a transactivation of the hsp70 promoter is mediated by CCAAT binding factor (CBF) . It is demonstrated here that p53 both represses transcription from the human hsp70 promoter and also interacts with CBF . Thus, the repression of the hsp70 promoter by p53 may be mediated by direct protein-protein interaction with CBF . These results suggest that protein-protein interaction between p53 and specific transcription factors may be an additional mechanism by which p53 regulates gene expression. Scand J Immunol, 1993 Jan, 37(1), 29 - 32 Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and rhTNF-alpha analogue enhance amyloid deposition in the Syrian hamster; Niewold TA et al.; Tumour necrosis factor-alpha (TNF-alpha) is one of the cytokines that stimulate the production of serum amyloid A (SAA), the precursor of AA amyloid . The role of TNF-alpha in amyloidogenesis was investigated in experimental hamsters using purified recombinant human TNF-alpha (rhTNF-alpha) and rhTNF-alpha analogue different from the normal molecule by two amino acid substitutions . Daily injections of 1 microgram rhTNF-alpha resulted in elevated SAA levels but even in the presence of amyloid enhancing factor (AEF) no amyloid was deposited, indicating that apart from the AEF and one particular SAA stimulating factor an additional factor is needed to result in amyloid deposition . This factor is generated by repeated injections of E . coli lipopolysaccharide (LPS) . A single intraperitoneal injection of 12.5 micrograms or more of rhTNF-alpha followed by seven daily subcutaneous injections of LPS resulted in enhanced amyloid deposition . Heat denaturation of rhTNF-alpha did abolish its AEF activity . The rhTNF-alpha analogue, having one-fifth of the cytotoxic activity of the normal rhTNF-alpha, showed a similar reduction in its SAA-inducing capacity and its amyloidogenicity . This suggests the AEF activity to be closely related to TNF-alpha activity . However, poly(I)-poly(C) (a potent inducer of IL-6) also showed AEF activity, suggesting that not a single cytokine but rather a certain combination of different cytokines could be decisive in AA amyloidogenesis. Leukemia, 1993 Jan, 7(1), 131 - 9 Retrovirus-mediated transfer and expression of marker genes in the BN rat acute myelocytic leukemia model for the study of minimal residual disease (MRD); Yan Y et al.; To study minimal residual disease (MRD) in leukemia, we transferred the Escherichia coli genes encoding beta-galactosidase (lacZ) and neomycin resistance (neo(r)) into the subline LT12 of the Brown Norway rat acute myelocytic leukemia (BNML), employing the retroviral BAG vector . In this way leukemic cells were genetically marked . Ten independent cell lines were characterized during in vitro growth as well as during two subsequent in vivo passages for expression of neo(r) for which the neomycin analogue G418 was used, and for lacZ expression for which the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used . Out of 10 lines, four revealed permanent high expression of lacZ in all cells . In four other lines greatly varying lacZ expression between the individual cells from these lines was observed . In the remaining two lines lacZ expression was gradually lost . In contrast, neo(r) expression was gradually lost in eight out of the 10 lines, particularly rapidly during in vivo passaging . In the remaining two lines neo(r) expression was retained . The genetic modification did not alter the in vitro leukemogenicity of the cells . Long term in vivo expression of neo(r) and lacZ was followed in two selected lines up to 12 subsequent passages, i.e . one from the group of homogeneous high lacZ expression and one from the group of heterogeneous lacZ expression . In both lines lacZ expression was retained whereas neo(r) expression was rapidly lost after the third passage . The feasibility of using genetically marked leukemic cells for studies of minimal residual disease (MRD) was explored by injecting rats with leukemic cells, treating them with chemotherapy at full blown leukemia development to reduce the tumor load, mimicking the induction of a state of MRD and studying lacZ expression at relapse . LacZ expression was evident in 100% of the cells whereas neo(r) expression was lost in a considerable fraction . These results indicate that the viral vector BAG can be used to mark leukemia cells genetically although a selection of clones with the desired stability of long-term expression is required. Infect Immun, 1993 Jan, 61(1), 81 - 90 Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA; Erdile LF et al.; OspA is a protective antigen of the Lyme disease spirochete Borrelia burgdorferi . Expression of the full-length B . burgdorferi B31 OspA gene in Escherichia coli produces a protein that is processed posttranslationally by signal peptidase II and contains an attached lipid moiety . The recombinant OspA lipoprotein has been purified by detergent extraction and ion-exchange chromatography . Priming and boosting with OspA lipoprotein, either with no adjuvant or adsorbed to alum, elicited a strong, dose-dependent immunoglobulin G response . Serum from vaccinated mice inhibited spirochetal growth in vitro . Mice immunized twice with as little as 0.4 micrograms of OspA lipoprotein were protected against an intradermal challenge with 10(4) infectious spirochetes . The ability of the purified recombinant lipoprotein to induce a strong protective response in the absence of toxic adjuvants makes it an excellent candidate antigen for a human vaccine against Lyme disease . By contrast, no serum immunoglobulin G or growth inhibitory response to OspA nonlipoprotein was seen at any dose . The difference in immunogenicities of the lipoprotein and nonlipoprotein forms of OspA is not due to any difference in the antigenicities of the two proteins . These results suggest that posttranslational lipid attachment is a critical determinant of the immunogenicity of the OspA protein. Genetics, 1993 Jan, 133(1), 29 - 38 Identification of Aspergillus brlA response elements (BREs) by genetic selection in yeast; Chang YC et al.; The brlA gene of Aspergillus nidulans plays a central role in controlling conidiophore development . To test the hypothesis that brlA encodes a transcriptional regulator and to identify sites of interaction for the BrlA polypeptide, we expressed brlA in Saccharomyces cerevisiae (yeast) strains containing Aspergillus DNA sequences inserted upstream of a minimal yeast promoter fused to the Escherichia coli lacZ gene . Initially, a DNA fragment from the promoter region of the developmentally regulated rodA gene was tested and shown to mediate brlA-dependent transcriptional activation . Two additional DNA fragments were selected from an Aspergillus genomic library by their ability to respond to brlA in yeast . These fragments contained multiple copies of a sequence motif present in the rodA fragment, which we propose to be sites for BrlA interaction and designate brlA response elements (BREs) . DNA fragments containing BREs upstream of a minimal Aspergillus promoter were capable of conferring developmental regulation in Aspergillus . Deletion of BREs from the upstream region of rodA greatly decreased its developmental induction . Multiple copies of a synthetic oligonucleotide with the consensus sequence identified among the BREs mediated brlA-dependent transcriptional activation in yeast . The results show that a primary activity of brlA is transcriptional activation and tentatively identify sites of interaction for the BrlA polypeptide. Mol Cell Biol, 1993 Jan, 13(1), 533 - 42 Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts; Carty MP et al.; We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189 . We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated . This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid . These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo . Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels . Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction . Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E . coli photolyase after replication but before T4 endonuclease treatment . These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers . Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism . All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation. J Clin Microbiol, 1993 Jan, 31(1), 9 - 15 Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli; van Gelder P et al.; A 330-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii 54-kDa rhoptry protein (ROP2) was expressed in Escherichia coli as a fusion polypeptide containing a 48-amino-acid sequence derived from phage lambda protein Cro and E . coli protein LacI followed by six consecutive histidyl residues . Metal chelate affinity chromatography provided an easy way to isolate the recombinant product in a highly purified form (> 95%) . When this material was used to develop an immunoglobulin G (IgG) enzyme-linked immunosorbent assay for diagnosis of toxoplasmosis, the test reached a sensitivity of 89% . The sensitivity of the assay was similar whether the sera contained T . gondii-specific IgM or were devoid of such IgM . It was also found that ROP2 is a conserved antigen since antibodies against the recombinant antigen could be detected in mice experimentally infected with 11 independent T . gondii isolates originating from infected human tissues tested . Thus, the 54-kDa rhoptry antigen could advantageously complement other previously described T . gondii antigens for the development of more-sensitive and more-informative recombinant antigen-based tests for toxoplasmosis diagnosis. J Cell Biol, 1993 Jan, 120(1), 95 - 102 Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70; Brodsky JL et al.; We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids . Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes . Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes . Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E . coli dnaK protein did not replace BiP . Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70 . We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane. J Bacteriol, 1993 Jan, 175(1), 74 - 9 Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli; Rao NN et al.; In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source . The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate . These cells were devoid of alkaline phosphatase activity . However, the phoA promoter was fully active in a glpT mutant . These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium. J Bacteriol, 1993 Jan, 175(1), 7 - 11 Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus; Park JT; Turnover of murein in oligopeptide permease-negative Escherichia coli cells appeared to be minimal or nonexistent . In one strain in which it was possible to measure turnover during the first generation of chase, it was found that the rate of turnover was constant throughout a chase of three generations . This result suggests that an "inside-to-outside" mode of growth of the sacculus does not occur in E . coli . Turnover, though minimal, was significantly higher from cells labeled uniformly than from cells labeled only in the lateral wall, suggesting that a significant portion of the observed turnover is related to cell separation . Actually, turnover only appeared to be minimal in opp mutant strains . Tripeptides were being released by turnover at a rate of about 50% per generation and then were efficiently recycled . This suggests that in addition to opp, a low-affinity uptake system for tripeptide derived from the sacculus may exist. J Bacteriol, 1993 Jan, 175(1), 53 - 63 Role of Clp protease subunits in degradation of carbon starvation proteins in Escherichia coli; Damerau K et al.; When deprived of a carbon source, Escherichia coli induces the synthesis of a group of carbon starvation proteins . The degradation of proteins labeled during starvation was found to be an energy-dependent process which was inhibited by the addition of KCN and accelerated when cells were resupplied with a carbon source . The degradation of the starvation proteins did not require the ATP-dependent Lon protease or the energy-independent proteases protease I, protease IV, OmpT, and DegP . During starvation, mutants lacking either the ClpA or ClpP subunit of the ATP-dependent Clp protease showed a partial reduction in the degradation of starvation proteins . Strains lacking ClpP failed to increase degradation of starvation proteins when glucose was added to starving cells . The clpP mutants showed a competitive disadvantage compared with wild-type cells when exposed to repeated cycles of carbon starvation and growth . Surprisingly, the glucose-stimulated, ClpP-dependent degradation of starvation proteins did not require either the ClpA or ClpB protein . The patterns of synthesis of starvation proteins were similar in clpP+ and clpP cells . The clpP mutants had reduced rates of degradation of certain starvation proteins in the membrane fraction when a carbon source was resupplied to the starved cells. J Bacteriol, 1993 Jan, 175(1), 303 - 6 Mutational uncoupling of the transcriptional activation function of the TyrR protein of Escherichia coli K-12 from the repression function; Cui J et al.; The tyrosine repressor (TyrR) protein of Escherichia coli can function either as a transcriptional enhancer or as a repressor . The structural basis for these opposite effects was analyzed in specific tyrR deletion mutants constructed in vitro . The functional behavior of the mutant TyrR proteins was evaluated in vivo by using single-copy lacZ reporter systems based on the mtr promoter (10-fold activation by wild-type TyrR protein, mediated by phenylalanine or tyrosine) or the aroF promoter (over 20-fold repression by wild-type TyrR protein, mediated by tyrosine) . A mutant TyrR protein lacking amino acids 2 to 9 was completely devoid of transcriptional activation function . Five additional mutant TyrR proteins lacking progressively greater numbers of N-terminal amino acids were likewise activation defective . The mutant TyrR proteins lacking amino acid residues 2 to 9 or 2 to 19 were essentially identical to the wild-type TyrR protein in their ability to repress the aroF promoter . Three other TyrR mutant proteins, lacking up to 143 amino acid residues from the N-terminal end of the protein, retained the ability to repress the aroF promoter, to different extents, in a tyrosine-dependent manner. J Bacteriol, 1993 Jan, 175(1), 259 - 65 Osmotic regulation of rpoS-dependent genes in Escherichia coli; Hengge-Aronis R et al.; The rpoS gene, which encodes a putative alternative sigma factor (sigma S), is essential for the expression of a variety of stationary-phase-induced genes as well as for stationary-phase-specific multiple-stress resistance . As previously shown for the otsA and otsB genes (R . Hengge-Aronis, W . Klein, R . Lange, M . Rimmele, and W . Boos, J . Bacteriol . 173:7918-7924, 1991), we demonstrate here that additional rpoS-controlled genes (bolA, csi-5) as well as at least 18 proteins on two-dimensional O'Farrell gels could be induced in growing cells by osmotic upshift via an rpoS-dependent mechanism . Also, rpoS-dependent thermotolerance and resistance against hydrogen peroxide could be osmotically stimulated . In contrast, the expression of glgS, while exhibiting strong stationary-phase induction, was only weakly increased by elevated osmolarity, and several rpoS-dependent proteins previously identified on two-dimensional gels were not osmotically induced . During osmotic induction of rpoS-dependent genes, rpoS transcription and the level of sigma S remained unchanged . We conclude that osmotically regulated genes represent a subfamily within the rpoS regulon that requires differential regulation in addition to that provided by sigma S. J Bacteriol, 1993 Jan, 175(1), 251 - 8 Control of transcription of gal repressor and isorepressor genes in Escherichia coli; Weickert MJ et al.; Two regulatory proteins, Gal repressor and isorepressor, control the expression of the gal and mgl operons in Escherichia coli . The transcription start sites for galR and galS, the genes for the repressor and isorepressor, were determined by primer extension of in vivo transcripts . Study of the promoter-lacZ gene fusions introduced into the chromosome indicated that galS expression was elevated in cells in which the normal galS gene was interrupted, but not in cells in which the galR gene was deleted . When both genes were disrupted, galS expression was further elevated . Expression from the galS promoter was stimulated by the addition of D-fucose, repressed by glucose, and dependent on cyclic AMP receptor protein (CRP) . Expression of a similar gene fusion of the galR promoter to lacZ was unregulated . Both galR and galS genes contain two potential operator sites (OE and OI) and a CRP-binding site . The arrangement of OE, OI, and the CRP-binding site in the galS gene is analogous to the arrangement in the gal and mgl promoters, but the arrangement in galR is atypical . The increased concentration of the isorepressor when inducer is present may facilitate early shutoff of the isorepressor-regulated genes of the gal regulon when inducer (substrate) concentration falls. J Bacteriol, 1993 Jan, 175(1), 229 - 39 Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III; Babitzke P et al.; RNase III is an endonuclease involved in processing both rRNA and certain mRNAs . To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene . In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed . The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P . Gegenheimer, N . Watson, and D . Apirion, J . Biol . Chem . 252:3064-3073, 1977) . In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs . In addition, these results indicate that RNase III is probably not required for general mRNA decay . Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did . Possible explanations of these results are discussed. J Bacteriol, 1993 Jan, 175(1), 190 - 9 Effects of insertions and deletions in glnG (ntrC) of Escherichia coli on nitrogen regulator I-dependent DNA binding and transcriptional activation; Shiau SP et al.; Phosphorylated nitrogen regulator I (NRI, also called NTRC), encoded by glnG (ntrC), stimulates transcription in Escherichia coli and other enteric bacteria from sites analogous to eukaryotic enhancers . We isolated 30 mutants, obtained without phenotypic selection, that have either a small insertion or deletion within glnG . Mutants were classified by the ability of NRI to repress the glnAp1 and glnL promoters and to activate two versions of the nitrogen-regulated glnAp2 promoter; each activity was measured in cells grown with three concentrations of NRI . The results were interpreted within the framework of the three-domain hypothesis of NRI structure . NRI is thought to contain a phosphorylated regulatory domain that controls binding of ATP, a central domain that hydrolyzes ATP and interacts with RNA polymerase, and a DNA-binding region of unknown extent . Our results suggest that the 70 amino acids from residue 400 to the carboxyl terminus constitute a DNA-binding domain that includes residues for specific contacts and dimerization . Our results also suggest that (i) transcription from glnAp2 without specific NRI-binding sites requires binding to sites with some similarity to the specific sites, and (ii) if an NRI variant can stimulate transcription, then increasing the concentration of NRI diminishes glnA expression for all mutants but one. Dev Biol, 1993 Jan, 155(1), 161 - 71 Transient, localized accumulation of alpha-spectrin during sea urchin morphogenesis; Wessel GM et al.; The mRNA and protein of alpha-spectrin in the sea urchin embryo is shown here to be transiently overexpressed in cells initiating certain morphogenetic changes . This expression was detected by use of a 4.5-kb cDNA clone that encodes alpha-spectrin isolated from a Lytechinus variegatus cDNA library . DNA sequence analysis demonstrated 80% similarity of this cDNA to human nonerythroid alpha-spectrin . Sea urchin alpha-spectrin RNA accumulated to a uniform basal level in all cells of the embryo with a marked, transient increased accumulation in the primary mesenchyme cells of the vegetal plate just prior to gastrulation . Cells of the endoderm also express increased levels of spectrin during gastrulation and these levels remained high in the myoepithelial cells of the sphincter constrictions that separates the gut into foregut, midgut, and hindgut domains . Antibodies made to recombinant sea urchin spectrin synthesized in Escherichia coli were used to localize the protein in situ . These experiments showed that all cells of the embryo accumulated alpha-spectrin protein confined to the cortical regions of cell-cell contact . In epithelial cells, this localization showed a distinct honeycomb pattern . Primary mesenchyme cells and the myoepithelial cells of the gut sphincters showed significantly enhanced spectrin signal corresponding to the pronounced RNA localization pattern seen by in situ RNA hybridization . These data demonstrated that alpha-spectrin from the sea urchin is a highly conserved member of the spectrin superfamily of proteins and that its elevated expression in specific cell types anticipates overt morphogenesis. Exp Cell Res, 1993 Jan, 204(1), 61 - 72 Different subcellular localizations of discoidin I monomer and tetramer in Dictyostelium discoideum cells: using conformation-specific monoclonal antibodies; Fukuzawa M et al.; We characterized four monoclonal antibodies that recognize monomeric and/or tetrameric forms of Dictyostelium lectin (discoidin I) to study intracellular localization of this lectin in early development . Three different PAGEs (native-, urea-, and SDS-PAGE) following immunoblot showed that three of the four mAbs exhibit preference for the tetrameric form while mAb DC2 reacts only with unreduced monomer . By immunofluorescence studies of Dictyostelium NC-4, we found that the anti-tetramer antibodies mainly stain multilamellar bodies, which are food vacuoles to be externalized from cells . In contrast, mAb DC2 stains the cytosol weakly but not the multilamellar bodies . The same pattern of distribution was confirmed by immunoelectron microscopy . These results clearly indicate that, upon packaging discoidin I into multilamellar bodies, the tetrameric form is selectively packaged from the cytosolic pool . In addition, to clarify the relation between the multilamellar bodies and the tetramer, we examined the localization of tetramer using both the cells fed by Escherichia coli and the A3 cells axenically grown . When the cells were fed by E . coli, the cells made multilamellar bodies that contained no lectin; instead the tetramer was found in the cytoplasm and the cells still exclude the lectin around the cells . In contrast, A3 cells grown axenically without bacteria do not make multilamellar bodies but the tetramer also was found in the cytoplasm evenly (also, see W.F . Loomis, Dictyostelium Discoideum, pp . 160-161, Academic Press, New York, 1975) . The data suggest that, under these conditions, the tetramer can be excluded from cells via some organelle such as normal secretory vacuole but not multilamellar bodies . Moreover this represents the first example of differential localization of tetramer and monomer of discoidin I in the cellular slime mold cells. Cancer Res, 1993 Jan 1, 53(1), 176 - 82 Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ; Lampson LA et al.; We extend use of the lacZ reporter gene for tumor biology . Intracerebral growth of 9L/lacZ, a gliosarcoma cell line that stably expresses lacZ, was evaluated in syngeneic rats . The reporter gene product, Escherichia coli-derived beta-galactosidase (beta-gal), was detected histochemically on tissue sections . This permits visualization of disseminated tumor and, as shown here, facilitates image analysis . We show that the beta-gal marker protein itself can serve as a tumor antigen in appropriate contexts . Quantitative image analysis of tumor areas is used to show that immunization with beta-gal protects against tumor growth . Abnormal beta-gal- areas are easily detected, facilitating study of antigenic modulation . The tumor studied did not escape through this mechanism . All abnormal beta-gal- areas examined were shown to reflect accumulation of inflammatory or reactive cells, not tumor . Taken together, these findings show several ways in which the lacZ reporter gene can be exploited to facilitate quantitative analysis of disseminated tumor growth within the brain . They draw attention to the growing appreciation that tumor antigens need not be cell surface molecules. J Virol, 1993 Jan, 67(1), 425 - 37 Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations; Vincent KA et al.; Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell . The integration reaction is mediated by integrase, a viral enzyme . Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity . Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates . Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration) . Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities . Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region . All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein . However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA . This hypothesis is supported by the results of disintegration assays performed with altered substrates . The results support a model involving separate viral and target DNA-binding sites on integrase. Khirurgiia (Sofiia), 1993, 46(1), 32 - 6 {Comparative studies of acid-base and gas metabolism in the whole blood and erythrocytes in endotoxic shock in rabbits}; Ianev E et al.; Parameters of acid-base and gas metabolism in whole blood and in red cell hemolysate were determined 30, 60 and 120 minutes after inducing endotoxin shock in rabbits of Belgian giant breed . Astrup's micromethod on apparatus of the firm "Radiometer" was used . Endotoxin shock was induced by intravenous injection of 2 mg/kg endotoxin from E . coli 0111:B4 strain . Equations were used for expressing the relation between pH of erythrocytes and pH of whole blood and analysis made of the correlation between the changes in the individual components of erythrocyte and of whole blood acid-base metabolism . Severe metabolic acidosis developed both in whole arterial blood, in mixed venous blood and in red cell hemolysate . Close correlation was recorded between the changes in the parameters of acid-base metabolism in whole blood and in red cell hemolysate on the 60 . and especially on the 120 . minute after endotoxin administration . The blood gas changes manifested by slight decrease of PCO2 and PO2 both in arterial and in mixed venous whole blood and red cell hemolysate were not statistically significant . The severe tissue hypoxia during the early phases of endotoxin shock was thought to be the result of severe hemodynamic changes. Cytobios, 1993, 75(301), 69 - 84 Origins of spontaneous mutants: cellular strategies for controlling their formation in response to changing environments; MacPhee DG; Spontaneous and induced mutational events may not be as readily distinguishable as has been generally assumed . It is suggested that the later stages of the processes leading to the establishment of fixation of mutations may consist of a 'mutational pathway', with many features in common with the better-known metabolic pathways of organisms like Escherichia coli . If so, the mutational pathway may be controlled by catabolite repression, a cellular mechanism which switches metabolic pathways on and off (or more probably up and down) in response to intracellular levels of a molecular messenger known as cyclic adenosine monophosphate (cAMP) . Mutation rates could then be regulated by an intracellular messenger in precisely the way which we would predict they ought to be regulated, if they are to cope with evolutionary pressures for increased variability. Surg Today, 1993, 23(8), 747 - 9 Gas-forming liver abscess after transcatheter arterial embolization for hepatocellular carcinoma: report of a case; Hanazaki K et al.; A case of a gas-forming liver abscess developing after transcatheter arterial embolization for recurrent hepatocellular carcinoma (HCC) in a 65-year-old man is presented herein . He was admitted to hospital with fever and jaundice, following which ultrasonography (US) and computed tomography revealed a gas-containing abscess in the posterior segment of the hepatic lobe with multiple HCC . Percutaneous transhepatic drainage was performed using US . Antibiotics which were sensitive to the Escherichia coli bacteria detected in the abscess were administered both intravenously and through the drainage tube into the abscess . Four months later, the abscess had diminished and the patient was discharged after receiving percutaneous ultrasonographically guided ethanol injection therapy for the recurrent HCC. Arch Microbiol, 1993, 160(2), 81 - 6 Effect of novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), on the osmoprotectant activity of glycine betaine, choline and L-proline in Escherichia coli; Kunin CM et al.; A novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), was found to block the osmoprotectant activity of choline and L-proline, but not glycine betaine in Escherichia coli . MPMS was more active against salt-sensitive than salt-resistant strains, but had no effect on the salt tolerance of a mutant which was unable to transport choline, glycine betaine and proline . Growth of E . coli in NaCl was inhibited by MPMS and restored by glycine betaine, but not by choline or L-proline . Uptake of radiolabeled glycine betaine, choline or L-proline by cells grown at high osmolarity was not inhibited when MPMS and the radioactive substrates were added simultaneously . Preincubation for 5 min with MPMS reduced the uptake of choline and L-proline, but not glycine betaine . Similar incubation with MPMS had no effect on the uptake of radiolabeled glucose or succinate . The toxicity of MPMS was much lower than that of the L-proline analogues L-azetidine-2-carboxylic acid and 3,4-dehydro-DL-proline . The exact mechanism by which MPMS exerts its effect is not entirely clear . MPMS or a metabolite may interfere with the activity of several independent permeases involved in the uptake of osmoprotective compounds, or the conversion of choline to glycine betaine, or effect the expression of some of the osmoregulatory genes. Vaccine, 1993, 11(8), 825 - 9 Protection of swine against foot-and-mouth disease with viral capsid proteins expressed in heterologous systems; Grubman MJ et al.; Three groups of swine were each inoculated with a different antigen preparation of foot-and-mouth disease virus (FMDV) capsid proteins and challenged by contact exposure to animals infected with FMDV . One group of four animals was inoculated with an extract from cells infected with a recombinant baculovirus containing the FMDV P1-2A structural protein precursor gene and a portion of the P2 gene . Two out of four animals were protected from clinical disease, but not from virus replication . A second group of animals was inoculated with an extract from Escherichia coli that expressed FMDV proteins from a construct containing the P1-2A gene, a portion of the P2 gene and the 3C protease gene . Three out of four animals in this group did not develop clinical signs of FMD upon challenge and two of four were protected against virus replication . In contrast, inoculation of a third group of swine with an extract from E . coli expressing the same FMDV construct as present in the recombinant baculovirus failed to protect any of the four animals from generalized FMD. Arch Virol, 1993, 131(3-4), 431 - 9 Role of the TK+ phenotype in the stability of pigeonpox virus recombinant; Letellier C; Insertion of foreign DNA containing the E . coli gpt marker by homologous recombination in the pigeonpox virus (PPV) thymidine kinase (TK) gene and selection for the presence of this DNA in the viral genome produced unstable recombinants after 3 plaque purifications . We highlight the persistence of duplicated TK DNA sequences arising from single crossing over, due to the growth advantage of TK+ virus . Restoration of the TK function by coinsertion of the vaccinia virus TK gene led to stable TK+ recombinants arising from double crossing over. J Comp Physiol {B}, 1993, 163(2), 89 - 98 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties; Pyko M et al.; Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues . This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise . 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques . 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold . 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional . The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis . The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme . It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase . The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300) . The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5 . However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme . 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect . Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS) Vet Med (Praha), 1993, 38(4), 237 - 44 {Comparison of various antigens in the diagnosis of caprine arthritis-encephalitis virus using the ELISA test}; Celer V Jr et al.; The most adequate means of diagnosing infections with caprine arthritis encephalitis (CAE) and Maedi-Visna (MV) viruses is the demonstration of antibodies to these viruses in milk or in blood serum . Various techniques and a range of different antigen preparations can be used for this purpose . We have evaluated two different whole virus antigen preparations derived from lamb synovial cells infected with CAE and MV viruses and recombinant antigen containing the capsid protein expressed in E . coli in ELISA test . The suitability of different detergents for solubilization of whole virus particles is shown in Tab . I . The detergents used were ether (50%, 10 min, 37 degrees C), octyl-beta-D glucopyranoside - OGP (2%, 2 h, 37 degrees C), and sodium dodecylsulphate SDS (0.125%, 10 min, 37 degrees C) . Antigens prepared with SDS gave the best results and were then used in the following antigen comparative study . All antigens (whole virus and recombinant core protein) were used for the coating of ELISA plates and were evaluated on a panel of 130 sera . In general, whole virus antigens prepared from CAE- and MV-viruses gave identical results . The specificity achieved with recombinant antigen was superior but the sensitivity was lower compared with whole virus antigen . Immunoblotting served as the gold standard for discordant results . The other aim of our study was to compare antibody detection in blood serum and milk . Using whole virus antigen, antibodies could be detected with higher specificity and sensitivity in milk than in blood serum (Tab . II).(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Endocrinol, 1993 Jan, 90(2), R11 - 6 Interaction of androgen and glucocorticoid receptor DNA-binding domains with their response elements; De Vos P et al.; Fusion proteins containing the glucocorticoid and the androgen receptor DNA-binding domain (ARF1 and GRF1) were produced in Escherichia coli . DNAse I footprinting was used to compare the interaction of these proteins with responsive elements (REs) in a typically glucocorticoid-responsive gene (mouse mammary tumour virus (MMTV)) and in an androgen-responsive gene (the C3(1) gene of rat prostatic binding protein) . It is demonstrated that response elements which most closely resemble the consensus sequence show identical footprinting patterns for ARF1 and GRF1 . The protected regions suggest that these sequences are occupied by two DNA-binding domains (DBDs) forming a dimer . Regions that constitute imperfect RE sequences, however, are apparently recognized by only one DBD, which mainly protects the TGTTCT motif . At these REs, the protection patterns produced by ARF1 and GRF1 are not identical . In the long terminal repeat (LTR) of MMTV but not in C3(1), a mechanism other than classical dimer formation seems to increase the affinity of ARF1 and GFR1 for these imperfect REs. Circ Shock, 1993 Jan, 39(1), 44 - 51 Prolonged lung edema formation during the generalized Shwartzman reaction in rabbits--can it be a model for septic lung disease? Miyata T, Torisu M, Toh H, Goya T. Pulmonary edema formation and the role of polymorphonuclear leukocytes (PMN) was evaluated during the generalized Shwartzman reaction (GSR) model in rabbits . Chemiluminescence (CL) and superoxide (O2-) production activity stimulated by FMLP were measured in circulating PMN after a single intravenous injection of endotoxin (E . coli 026:B6, 40 micrograms/kg) (E1) . CL peaked at 36 hr after E1 injection and then decreased gradually to a normal range within 7 days . O2- production changed in a similar fashion . On the other hand, the PMN count peaked at 72 hr and remained at a high value until day 7 . To induce GSR, a second venous injection of endotoxin (40 micrograms/kg) (E2) was administered 36 hr after the first one (E1) . Four times as many PMN were observed in the lung capillaries 4 hr after the second endotoxin injection than after the first endotoxin injection (4 hr) . The degree of lung edema formed increased after E2, but did not increase after E1 . An alveolar hyaline-like exudate was observed 24 hr after E2 . A continuous intravenous injection of superoxide dismutase (SOD) plus catalase or PMN depletion, prevented the development of post-E2 lung edema . If E2 was given 7 days after E1, no lung edema formed at all . These data indicate that oxygen free radicals from activated PMN by endotoxin play an important role in prolonged lung edema formation in the GSR model. Zhonghua Yi Xue Za Zhi (Taipei), 1993 Jan, 51(1), 33 - 9 Hepatolithiasis, a clinical study; Jiang CF et al.; Ninety-seven hepatolithiasis cases were reviewed retrospectively . Primary hepatolithiasis was about equal in both sexes, with most patients under 39 years of age . Most secondary hepatolithiasis patients who were female-predominant, were older than 40 . Common presenting symptoms were abdominal pain, either epigastric or right upper quadrant of abdomen as noted in 93%; chills and fever in 70%; nausea and vomiting in 49.5%; jaundice was noted only in 39% of the patients . Blood tests showed elevation of alkaline phosphatase in 79.8%, and gamma-GT in 88.3% . Left branch involvement was much more common that right . Escherichia coli (E . coli) was the main organism isolated in most of the cases . Abdominal ultrasonography reached a diagnostic rate of 91.01%, and the condition could be missed in cases of intrahepatic muddy stones, pneumobilia and misidentification of the location of the stones . Endoscopic retrograde cholangiography (ERC) showed a clearer picture of the biliary tree, but failed in cases of distorted anatomy because of previous operation, stones impacted in the ampulla orifice, presence of diverticulum or poor opacification of the bile duct because of stricture or stone impaction. Avian Dis, 1993 Jan-Mar, 37(1), 1 - 5 Use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys; Newberry LA et al.; Three hundred fifty 1-day-old large white turkeys were reared in brooding batteries to 10 days of age, after which they were moved to floor pens on litter . At 7 weeks of age, poults were allotted into four treatment groups as follows: 1) virulent hemorrhagic enteritis virus (HEV) alone (100 turkeys), 2) Escherichia coli alone (100 turkeys), 3) HEV + E . coli (100 turkeys), and 4) negative controls (50 turkeys) . HEV was given orally at 7 weeks of age, followed by E . coli challenge in the drinking water 2 days later for 10 consecutive days . All groups were observed daily for mortality, both during and after challenge . Turkeys that died or were moribund were necropsied, and cultures were taken from the liver and bone marrow for bacterial isolation . Total mortality rates were 23% in the HEV + E . coli group, 10% in the HEV-only group, 3% in the E . coli-only group, and 0% in the negative control group . Cumulative mortality values were significantly different from those of the negative controls (P < or = 0.05) for HEV only and the HEV + E . coli group . E . coli was isolated from the liver and bone marrow of almost all turkeys that died. Mol Gen Genet, 1993 Jan, 236(2-3), 267 - 74 DNA binding domains in Tn3 transposase; Maekawa T et al.; Various segments of Tn3 transposase were fused individually to beta-galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay . Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1-242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243-632) had non-specific DNA binding ability . Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1-86 and 87-242), neither of which had specific DNA binding ability, but which both possessed non-specific DNA binding ability . The central segment included two subsegments (amino acid positions 243-289 and 439-505) with non-specific DNA binding ability . These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity . The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability . This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition. J Cell Biochem, 1993 Jan, 51(1), 14 - 8 The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology; LaPorte DC; Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation . This phosphorylation cycle controls the flow of isocitrate through the glyoxylate bypass, a pathway which bypasses the CO2 evolving steps of the Krebs' cycle . IDH is phosphorylated at a single serine which resides in its active site . Phosphorylation blocks isocitrate binding, thereby inactivating IDH . The IDH phosphorylation cycle is catalyzed by a bifunctional protein kinase/phosphatase . The kinase and phosphatase reactions appear to be catalyzed at the same site and may share some catalytic steps . A variety of approaches have been used to examine the IDH phosphorylation cycle in the intact organism . The picture which has emerged is one of an exquisitely sensitive and flexible system which is capable of adapting efficiently to the environment both inside and outside the cell. Antimicrob Agents Chemother, 1993 Jan, 37(1), 126 - 7 A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex; Willmott CJ et al.; Binding of the quinolone drug norfloxacin to gyrase and DNA has been investigated . We have detected binding to gyrase-DNA complex but find no significant binding to either gyrase or DNA alone . Enzyme containing gyrase A protein with the mutation Ser-83 to Trp (conferring quinolone resistance) showed greatly reduced drug binding. Am J Physiol, 1993 Jan, 264(1 Pt 1), G172 - 8 Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells; Mann EA et al.; Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals . The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase . Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors . Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity . Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor . Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies . However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity. Curr Genet, 1993 Jan, 23(1), 28 - 32 Development of a transformation system for the filamentous, ML-236B (compactin)--producing fungus Penicillium citrinum; Nara F et al.; We present here the first report of a transformation system developed for the filamentous, ML-236B (compactin)-producing fungus Penicillium citrinum . Hygromycin B-resistant colonies were obtained after treatment of protoplasts with a vector containing an Escherichia coli hygromycin B phosphotransferase gene fused to a 3-phosphoglycerate kinase promoter from Aspergillus nidulans . The transformation rate was 194 transformants per microgram circular DNA per 4 x 10(5) viable protoplasts under optimized transformation conditions . Transformation took place via the integration of plasmid DNA into the fungal chromosomal DNA . Most of the integration events appeared to produce tandemly iterated arrays of plasmid molecules at different sites in the chromosome . The transformed, drug-resistant, phenotype and the integrated plasmids were mitotically stable with or without selection in a majority of cases . The demonstration of such a transformation system is an essential first step in the application of recombinant DNA technology to strain improvement and for the production of novel ML-236B derivatives. Vaccine, 1993, 11(1), 25 - 35 Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV; van Drunen Littel-van den Hurk S et al.; High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems . The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves . With the exception of E . coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa . Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure . The E . coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies . This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine. Arch Biochem Biophys, 1993 Jan, 300(1), 510 - 6 Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b; Richardson TH et al.; Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha . The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E . coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript . The modified P450 2C3 was expressed at > 400 nmol/liter of culture . P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein . When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E . coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone . Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity . In addition, the enzyme expressed in E . coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone . These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone . Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E . coli for the expression of recombinant P450 enzymes. Arch Biochem Biophys, 1993 Jan, 300(1), 302 - 8 Effect of zinc removal on the conformation of Escherichia coli DNA topoisomerase I; Samuel M et al.; Escherichia coli DNA topoisomerase I contains three Zn(II) in each enzyme molecule required for relaxation of negatively supercoiled DNA . Apoenzymes were prepared from both the intact topoisomerase (M(r) 97,000) and the truncated active form top85 (M(r) 85,000) that lacks the carboxyl terminal domain but still contains the three Zn(II) . Fluorescence and circular dichroism spectroscopy were used to compare the apoenzymes with topoisomerase and top85 reconstituted with Zn2+ . The results indicated structural changes affecting the environment of the tryptophan residues and increasing the alpha-helical and beta-sheets content of the protein occurred upon zinc removal . These structural changes probably account for the loss of enzyme activity. J Gen Virol, 1993 Jan, 74 ( Pt 1), 55 - 64 Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions; Ogawa R et al.; Insertion of the Escherichia coli lacZ gene into a ClaI restriction enzyme site of a 5.7 kb HindIII fragment of the fowlpox virus (FPV) genome resulted in the generation of stable recombinants . These recombinants produced plaques that were significantly smaller than those produced by parental FPV or by FPV recombinants containing the lacZ gene at other non-essential sites . Insertion of foreign DNA into the ClaI site disrupts a previously unidentified open reading frame (ORF) which potentially encodes a 74K polypeptide . The predicted amino acid sequence of this FPV ORF has 24% identity with the F12L ORF of vaccinia virus, the function of which is not currently known . Production of intracellular FPV was similar in cells infected with recombinant or parental viruses, but the number of infectious extra