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PubMed2004

Association between up-regulation of stress-responsive genes and hypomethylation of genomic DNA in tobacco plants.

Transcripts that specifically accumulate in transgenic tobacco plants expressing an anti-sense construct for a tobacco type I DNA methyltransferase, NtMET1, were screened by the differential display method. Of the 31 genes identified, 16 encoded proteins with known functions; ten of these were related to biotic and abiotic stress responses, and the other six to cellular functions. In order to examine whether expression of these genes is correlated with DNA methylation status under natural stress conditions, a pathogen-responsive gene (NtAlix1) was selected as representative, and assayed for transcript induction and genomic methylation in tobacco plants infected with tobacco mosaic virus (TMV). In inoculated leaves of wild-type plants, NtAlix1 transcripts began to accumulate 12 h after the onset of the hypersensitive response (HR), and levels remained high for up to 24 h. Changes in the methylation status at the locus became obvious 24 h later, as detected by digestion of genomic DNA with a methylation-sensitive restriction enzyme. The results suggest that the level of DNA methylation may change in response to external stresses, and that this is closely related to the activation of stress-responsive genes.

Amino Acid Sequence↗
PubMed1998

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes.

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.

Amino Acid Sequence↗
PubMed2004

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly Bemisia tabaci.

To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) - a whitefly-transmitted geminivirus - was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.

Animals↗
PubMed1999

Genome evolution of tobacco mosaic virus populations during long-term passaging in a diverse range of hosts.

The effects of host changes on plant virus genome evolution was studied by nucleotide sequencing. A single tobacco (Nicotiana tabacum cv. Xanthi) plant was inoculated with in vitro transcripts from a plasmid clone of tobacco mosaic tobamovirus (TMV). This initial viral population was then transferred 11-12 times in parallel populations in 7 plant host species (1-4 replicates each) over a period of 413-515 days. Virion RNA was then isolated, reverse transcribed, amplified, cloned in bacteria, and sequenced. Portions of the coat protein, movement protein, and replicase genes were sequenced. Fourteen unique mutations were detected from a total of 188 clones (35,607 bases) sequenced, indicating a relatively small overall mutation rate of 3.1 x 10(-4) nucleotide substitutions/base-year. A small Ka/Ks value of 0.09 was also found, indicating selection against amino acid changes. Eighty-five percent of the substitutions were transitions. A G'(ST) value of 0.7 for the coat protein gene suggested that host type affected sequence changes in this region of the genome, but chi(2) analysis did not support this conclusion. This is the first study using sequencing to compare representative sample sections of a plant viral genome following a major selective disturbance such as extended passaging in an alternate host.

Genome, Viral↗
PubMed2000

Isolation and characterization of cDNAs encoding ribosome inactivating protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70-90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)+ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.

Amino Acid Sequence↗
PubMed1985

Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.

Animals↗
PubMed1983

Polymerization of tobacco mosaic virus protein without and with hydrogen ion binding.

When tobacco mosaic virus (TMV) protein is polymerized at pH values above 7 in unbuffered solutions, either by raising temperature at constant ionic strength or by increasing ionic strength at constant temperature, a 20 S component is formed having bound only the very small amount of H+ ion supplied by the unpolymerized protein. When hydrogen ion is added by titration during polymerization so as to keep pH constant, as would occur automatically if a buffer were present, a 20 S component is formed with one H+ ion bound each for half of the subunits. Thus, a 20 S form with and a 20 S form without bound H+ ion exist. Furthermore, the 20 S form without bound H+ ion binds H+ ion when supplied by titration to produce a 20 S form with the same amount of bound H+ ion as when H+ ion is supplied during the polymerization.

Hydrogen Bonding↗
PubMed1981

Tobacco mosaic virus protein induces fusion of liposome membranes.

The fusogenic properties of tobacco mosaic virus (TMV) coat protein were investigated. Tobacco mosaic virus protein induces membrane fusion of a population of L-alpha-dimyristoylphosphatidylcholine (DMPC) and DL-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles giving rise to larger particles as seen by a drastic absorbance increase of the liposomal solution. Differential scanning calorimetry spectra demonstrate complete mixing of the acyl chains of the lipids during fusion. Electron micrographs indicate that the fused entities are multilamellar.

Calorimetry, Differential Scanning↗
PubMed1979

Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5'-'cap' in viral messenger RNA.

Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tesetd in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m7G5' pppA that corresponds to the most simple 'cap' structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G5' pppA or G5' ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm7G). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pm7G carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pm7G was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the 'cap' component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addition of m7GpppA or pm7G when either TMV RNA or phage MS2 RNA was used as an mRNA.

Guanine Nucleotides↗
PubMed1990

Inhibition of protein synthesis in neoplastic cells by rhein.

The action of rhein, 4,5-dihydroxyanthraquinone-2-carboxylic acid, on protein synthesis of neoplastic cells has been investigated. Rhein decreases amino acid incorporation in all cells tested. The inhibition of incorporation of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake, which is unaffected by the drug. Tests on cell-free system showed that rhein does not inhibit the TMV-mRNA directed in vitro protein synthesis, thus indicating that the protein machinery per se is not affected. The inhibition of protein brought about by the drug must be ascribed to an effect on the energy-yielding processes with a remarkable decrease in ATP content. The mechanism is similar to that of other metabolic inhibitors, but rhein, for its capability to inhibit both respiration and glycolysis, is effective at much lower concentrations.

Adenosine Triphosphate↗
PubMed1990

Comparison of the enzymatic and edema-producing activities of two venom phospholipase A2 enzymes.

The edema-producing activity of NNAVPLA2, an acidic phospholipase A2 (PLA2) enzyme from Naja naja atra venom (NNAV), was less potent than that of TMVPLA2 II, a basic PLA2 from Trimeresurus mucrosquamatus venom (TMV). These edema-forming effects were greatly suppressed by pretreatment of rats with diphenhydramine/methysergide or compound 48/80, which reduced the tissue content of histamine and serotonin. Heparin abolished and suppressed the paw edema caused by protamine and TMVPLA2 II, respectively, but had no effect on the NNAVPLA2-induced response. In isolated rat peritoneal mast cells, both PLA2 concentration dependently induced the release of histamine and beta-glucuronidase. Again, TMVPLA2 II was more potent than NNAVPLA2. This degranulation effect of mast cells caused by TMVPLA2 II and protamine was inhibited by heparin, while that caused by NNAVPLA2 was unaffected. The edema-forming and mast cell degranulation effects were greatly decreased in both PBPB-modified NNAVPLA2 and PBPB-modified TMVPLA2 II, in which the catalytic activity of the enzymes was completely lost. PBPB-modified TMVPLA2 II-induced paw edema was also suppressed by heparin. Furthermore, this edematous response was totally reversed in rat pretreated with aspirin in combination with diphenhydramine and methysergide. These results suggest that the edema-forming effect of PLA2 is probably dependent on the presence of catalytic, positive charge and pharmacological sites on its molecule.

Animals↗
PubMed1985

Effect of lonidamine on protein synthesis in neoplastic cells.

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Amino Acids↗
PubMed1992

The 5' untranslated region from pea seedborne mosaic potyvirus RNA as a translational enhancer in pea and tobacco protoplasts.

We have exploited the transient expression of foreign genes introduced into plant protoplasts to investigate the effect of the pea seedborne mosaic potyvirus (PSbMV) 5' untranslated region (5'UTR) on the level of gene expression in pea and tobacco protoplasts. The plant viral 5'UTRs were found to increase translation significantly in comparison to a plasmid containing no 5'UTR of viral origin. The enhancement effect of the 5'UTRs of PSbMV and tobacco etch potyvirus (TEV) was found to be similar in pea and tobacco protoplasts, indicating a host-independent role of the potyviral 5'UTRs in enhancing gene expression. Translational enhancement of the two potyviral 5'UTRs was similar to that of the 5'UTR of tobacco mosaic virus (TMV). This observation makes it attractive to use potyviral 5'UTRs as general translational enhancers in future genetic transformations of plants.

Base Sequence↗
PubMed1994

Pyrimidine tract binding protein strongly stimulates in vitro encephalomyocarditis virus RNA translation at the level of preinitiation complex formation.

Cellular protein p57/58, now known to be identical to polypyrimidine tract binding protein (PTB), has earlier been shown to specifically bind to the internal ribosome entry sites (IRES) of encephalomyocarditis virus (EMCV) and some other picornaviral RNAs. To elucidate its relevance to the internal initiation, the effect of cloned purified PTB on EMCV IRES directed translation was studied in cytoplasmic extracts of Krebs-2 ascites carcinoma cells partially depleted of endogenous PTB. Addition of PTB to such extracts resulted in a strong stimulation of translation of a beta-glucuronidase (GUS) reporter cistron fused to the EMCV IRES, but had no effect on translation of capped mRNAs, such as beta-globin, and tobacco mosaic virus (TMV) RNAs. PTB was found to exert its effect at the level of 48S pre-initiation complex formation.

DNA-Binding Proteins↗
PubMed1994

Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions.

In an attempt to understand how Escherichia coli ribosomes recognize the initiator codon on mRNAs lacking the Shine-Dalgarno (SD) sequence, we have studied 30S initiation complex formation in extension inhibition (toeprinting) experiments using (-SD)mRNAs which are known to be reliably translated in E. coli: the plant viral messenger A1MV RNA 4 and two chimaeric mRNAs coding for beta-glucuronidase (GUS) and bearing the 5'-untranslated sequence of TMV RNA (omega) or the omega-derived sequence (CAA)n as 5'-leaders. Ribosomal protein S1 and IF3 have been found to be indispensable for translational initiation. Protein S1 appears to be a key recognition element. S1 binds to sequences within the leaders of (-SD)mRNAs thus providing their affinity to E. coli ribosomes.

Alfalfa mosaic virus↗
PubMed1988

A high-yield cell-free system of protein synthesis of mouse liver.

1. A fractionated cell-free system of protein synthesis has been developed from mouse liver. It is composed of polysomes, "pH 5" fraction, Mg2+, K+, ATP and a ATP generating system. 2. It operates optimally at 30-37 degrees C, in the presence of 4 mM MgCl2 and 90 mM KCl. 3. Spermine is highly inhibitory, while spermidine shows a bimodal action, in that submillimolar concentrations stimulate, while millimolar concentrations inhibit protein synthesis. 4. Both spermine and spermidine show an interesting selectivity, in that, even though they inhibit incorporation of amino acids into most proteins, they stimulate incorporation into a few proteins. 5. The system can be rendered mRNA-dependent, either by preincubation or by treatment with micrococcal nuclease. In both cases globin mRNA as well as TMV RNA are faithfully translated. 6. Compared to other published mammalian fractionated cell-free systems, the mouse liver system is more efficient by approximately one order of magnitude, since the rate of incorporation of leucine per min is 30 pmol/mg protein or 435 pmol/mg RNA or 1 mol/mol ribosomes.

Animals↗
PubMed1991

Measurement of affinity of viral monoclonal antibodies by ELISA titration of free antibody in equilibrium mixtures.

The binding affinity of a monoclonal antibody (Mab) to tobacco mosaic virus (TMV) was determined by measuring, in an enzyme-linked immunosorbent assay, the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. In antibody excess, univalent binding of Mabs was observed and the affinity constant was K = 3.2 +/- 0.4 10(8) l/mol; in antigen excess, bivalent antibody binding was observed and the antibody avidity was about 15 times higher. In antigen excess, it was imperative to correct experimental data for the presence of 0.55% inactive molecules in the immunopurified antibody preparation. Modelling studies suggest that in the case of antibodies of increasing affinity, it becomes increasingly important to correct for the presence of inactive antibody in the binding assay.

Antibodies, Monoclonal↗