Biomedical subjects
J Chappell
Publications and source records attributed to J Chappell.
Pre-steady-state study of recombinant sesquiterpene cyclases.
An Escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimeric 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein. From initial velocity experiments, the Michaelis constant for HVS was 3.5 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, respectively. Steady-state catalytic constants were from 0.02 to 0.04 s-1. A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in farnesyl diphosphate (FPP). Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step. Dissociation constants for the enzyme-substrate complex (20-70 microM) were determined from the pre-steady-state experiments and were significantly larger than the observed Michaelis constants. A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes. Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents.
Malignant cerebral glioma. A relative's perspective.
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Regulation of sesquiterpene cyclase gene expression. Characterization of an elicitor- and pathogen-inducible promoter.
The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the beta-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.
An analysis of clock-shift experiments: is scatter increased and deflection reduced in clock-shifted homing pigeons?
Clock-shifting (altering the phase of the internal clock) in homing pigeons leads to a deflection in the vanishing bearing of the clock-shifted group relative to controls. However, two unexplained phenomena are common in clock-shift experiments: the vanishing bearings of the clock-shifted group are often more scattered (with a shorter vector length) than those of the control group, and the deflection of the mean bearing of the clock-shifted group from that of the controls is often smaller than expected theoretically. Here, an analysis of 55 clock-shift experiments performed in four countries over 21 years is reported. The bearings of the clock-shifted groups were significantly more scattered than those of controls and less deflected than expected, but these effects were not significantly different at familiar and unfamiliar sites. The possible causes of the effects are discussed and evaluated with reference to this analysis and other experiments. The most likely causes appear to be conflict between the directions indicated by the sun compass and either unshifted familiar visual landmarks (at familiar sites only) or the unshifted magnetic compass (possible at both familiar and unfamiliar sites).
Identifying functional domains within terpene cyclases using a domain-swapping strategy.
Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes. Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction products of the Hyoscyamus enzyme. Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes. The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.
Conflict of ethics and practice in hospitalized patients who want to smoke.
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Erratum and apology re: "Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis".
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Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases.
Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.
Accurate in vitro transcription from circularized plasmid templates by plant whole cell extracts.
A convenient in vitro transcription system using monocot and dicot whole cell extracts and circular DNA templates has been developed. The system consists of incubating template and whole cell extract to generate initiation complexes, followed by addition of nucleotide triphosphates to support elongation, and primer extension assay to detect authentic transcripts. This in vitro transcription system required circularized templates and was essentially inactive with linearized templates. Accurate in vitro transcription of a rice phenylalanine ammonia-lyase (PAL) promoter-beta-glucuronidase (GUS) gene fusion and a tobacco sesquiterpene cyclase promoter-GUS gene fusion was examined in their homologous whole cell extracts, and the optimal concentrations for several reaction components, including DNA template, whole cell extract, monovalent and divalent cations, were determined for specific initiation from the in vivo start site. Transcription was inhibited by low concentrations of alpha-amanitin, demonstrating that the reaction was mediated by RNA polymerase II. Accurate transcription initiation was dependent on the TATA-box motif within the respective promoters. Based on the effect of delayed addition of sarkosyl at a concentration sufficient to inhibit transcription initiation but not elongation, three to four rounds of transcription were initiated in standard assays.
Expression of a plant sesquiterpene cyclase gene in Escherichia coli.
5-Epi-aristolochene synthase is a sesquiterpene cyclase activity found in pathogen-challenged tobacco cells, but not in nonchallenged tissues, and appears to be encoded by a complex gene family. As a prerequisite to assessing the functional significance of these multiple genes, bacterial expression systems were examined for their capacity to express a tobacco sesquiterpene cyclase cDNA. Insertion of full-length 5-epi-aristolochene synthase cDNA into two commonly used expression vectors, pET-11d and pGBT-T19, resulted in high level expression of the cyclase activity. The highest level of expression occurred 3 h after induction with low concentrations (0.1-0.5 mM) of IPTG, incubation at 27 degrees C instead of 37 degrees C, and in the bacterial host strain BL21(DE3). Under these conditions, the cyclase protein constituted 5 to 8% of the soluble and 35% of the total Escherichia coli proteins. Enzyme reaction products of the native tobacco and recombinant enzyme were identical, based on argentation-thin layer chromatography. Deletion mutants of the cyclase gene corresponding to the amino and carboxy termini of the enzyme were prepared. The cyclase proteins resulting from bacterial expression of these mutant constructs were found largely in the insoluble protein fraction and no soluble enzyme activity was detected.
Gene family for an elicitor-induced sesquiterpene cyclase in tobacco.
The initial step in the conversion of the isoprenoid intermediate farnesyl diphosphate to the sesquiterpenoid phytoalexin capsidiol in elicitor-treated tobacco tissues is catalyzed by an inducible sesquiterpene cyclase [5-epi-aristolochene synthase (EAS)]. Two independent cDNA clones (cEAS1 and cEAS2) encoding EAS were isolated from an elicitor-induced tobacco cDNA library by differential hybridization and subsequently were characterized by hybrid selection--in vitro translation. Insertion of cEAS1, a partial cDNA clone encoding 175 C-terminal amino acids, into an Escherichia coli expression vector resulted in accumulation of a fusion protein immunodetectable with EAS-specific polyclonal antibodies. The cDNA clones were used to isolate two full-length EAS genes that mapped 5 kilobases (kb) apart on one 15-kb genomic clone. The nucleotide sequences of the structural gene components were identical from 388 base pairs (bp) upstream of the transcription initiation site to 40 bp downstream of the translation termination codon, suggesting a relatively recent duplication event. The genes consist of 1479-bp open reading frames, each containing five introns and specifying 56,828-Da proteins. The N-terminal amino acid sequence deduced from the genomic clones was identical to the first 16 amino acids of the EAS protein identifiable by Edman degradation. RNA blot hybridization with cEAS1 demonstrated a mRNA induction time course consistent with the induction of the EAS mRNA translational activity with maximum levels 4-6 h after elicitation. EAS mRNA was not detected in control cells. DNA blot-hybridization analysis of genomic DNA revealed a copy number of approximately 12-15 for EAS-like genes in the tetraploid tobacco genome. The conservation of a putative allelic prenyl diphosphate binding motif is also discussed.
Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists.
A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [(35)S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.
Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.
Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.
Inhibition of a plant sesquiterpene cyclase by mevinolin.
The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.
Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures.
Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [(14)C]acetate and [(3)H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation.
Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures.
An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a K(m) for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.
Regulation of a sesquiterpene cyclase in cellulase-treated tobacco cell suspension cultures.
The regulation of an elicitor-inducible sesquiterpene cyclase in tobacco (Nicotiana tabacum) cell suspension cultures was investigated. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 15 hours of cellulase addition to the cell cultures. The induction of the cyclase activity was correlated with an absolute amount of the cyclase protein as measured in immunoblots. Both the in vivo synthesis rate, measured as the incorporation of [(35)S]methionine by cell cultures into immunoprecipitable cyclase protein, and the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of isolated RNA, were maximal at that time when the increase in cyclase enzyme activity was maximal. Using thiouridine to selectively label and isolate de novo synthesized mRNA, the in vitro translation products encoded by the newly synthesized RNA from elicitor-treated, but not control, cell cultures contained immunoprecipitable cyclase protein. These results suggest that the induction of the sesquiterpene cyclase in elicitor-treated cell cultures is primarily regulated by transcriptional control of the cyclase gene.