PubMed Health⌕ Search

Biomedical subjects

M E Tousignant

Publications and source records attributed to M E Tousignant.

At least 19 recordsLinked to original sources
PubMed2000

Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus.

To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome component RNA 3 of the virus was modified and split into two sub-components, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (MP) was replaced by a reporter gene encoding the green fluorescent protein (GFP), to monitor virus replication and movement. In RNA 3B, the coat protein (CP) gene was eliminated and a multiple cloning site (MCS) was created for foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector system was evaluated for its ability to deliver and express the bacterial beta-glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat protein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated leaf and enter the minor veins of the inoculated leaf. Foreign gene expression was detected in the inoculated leaves. However, intermolecular recombination between RNA 3A and 3B occurred frequently, preventing efficient systemic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.

Capsid↗
PubMed1995

The replication of a necrogenic cucumber mosaic virus satellite is temperature-sensitive in tomato.

Lethal necrosis development in tomato plants infected with cucumber mosaic virus (CMV) strain D containing the necrogenic satellite D-CARNA 5 and held at 32 degrees C is shown to be impaired. CARNA 5 accumulation in tomato at 32 degrees C is reduced about 100-fold compared to accumulation in plants held at 24 degrees C, while viral RNA accumulation is reduced about 5-fold. CMV-infected tomato held for 3 days at 24 degrees C prior to shift to 32 degrees C do not develop lethal necrosis. Longer incubations at 24 degrees C prior to shift to 32 degrees C allow necrosis to develop. CMV-infected plants held for up to 4 weeks at 32 degrees C required an additional 8-10 days at 24 degrees C to develop necrosis. Necrogenic CMV-infected plants held at 24 degrees C and analyzed 3 days p.i. contained detectable amounts of ss- and ds-CARNA 5; upon shift to 32 degrees C, such CARNA 5 declined to undetectable levels and lethal necrosis did not occur. There appear to be temperature-sensitive factors that are required for efficient satellite replication which are not required for efficient viral RNA replication. Whether these factor(s) are of host or satellite origin is uncertain.

Cucumovirus↗
PubMed1995

Effect of temperature on cucumber mosaic virus satellite-induced lethal tomato necrosis is helper virus strain dependent.

The effect of temperature on the response of tomato (Lycopersicon esculentum Mill. cv. Rutgers) to infections with the necrogenic cucumber mosaic virus (CMV) satellite D-CARNA 5 was investigated with each of four CMV strains D, 1, Y and S functioning as helper virus. At 24 degrees C lethal necrosis was observed in all infections. However, at 32 degrees C the response varied from total absence or reduction of necrosis with some strains to accelerated lethal necrosis with others. The total lack of necrotic response with CMV-S and the aggravated necrosis with CMV-Y at the higher temperature both turned out to be independent of the coinfecting satellite, and rather to correlate with the changing rate of viral RNA accumulation in tomato, which probably was responsible for the changes in pathogenic response. However, when CMV-D was helper virus, satellite accumulation decreased, while with CMV-1 it increased, respectively, while viral RNA accumulations were not seriously affected. Although these profound effects of temperature seem to link the necrotic response of tomato to the competitive replication dynamics of the infecting virus/satellite combination in the case of CMV-D/D-CARNA 5, temperature effects at other levels of disease induction probably play an important role as well.

Cucumovirus↗
PubMed1995

Small satellite of arabis mosaic virus: autolytic processing of in vitro transcripts of (+) and (-) polarity and infectivity of (+) strand transcripts.

In vitro RNA transcripts of both (+) and (-) polarities were obtained from a circularly permuted dimer clone of the small satellite of arabis mosaic virus (sArMV). The transcripts show efficient self-cleavage at the two specific sites in the sequence where the monomers were joined. Autolytic processing of the full-length transcript in both orientations releases promoter-proximate fragments (+) or (-) P, promoter-distal fragments (+) or (-) D, and the monomer fragments (+) or (-) M. The presence of an OH group at their 5' ends and a 2',3' cyclophosphate at their 3' ends suggests that (+) and (-) M originated via two self-cleavage reactions within the full-length transcript of corresponding polarity. Infectivity assays showed that the (+) M fragment but not the (-) M fragment initiates replication as efficiently as the natural linear sArMV in Chenopodium quinoa. Two (-) fragments were identified which are the result of religation activity: a P-D fragment formed by religation of P and D, and c-M, which is the result of efficient self-ligation of (-) M. In contrast, linear (+) M self-ligates in vitro to a very limited extent but could be circularized enzymically in a wheat germ extract.

Base Sequence↗
PubMed1993

Tomato necrosis and the 369 nucleotide Y satellite of cucumber mosaic virus: factors affecting satellite biological expression.

To determine which factors can affect biological expression of the Y satellite RNA of cucumber mosaic virus (CMV) in tomato, three laboratories collaboratively exchanged their natural satellite variants, the corresponding recombinant DNA clones and helper virus strains, as well as tomato varieties, on which different observations previously reported were based. The effects of these materials and the influence of temperature on symptom expression were systematically studied. The results show that in a standardized tomato bioassay at 24 degrees C, the Y satellite, when supported by either CMV-1 or CMV-Y, did not induce tomato necrosis in the Rutgers variety but elicited a slower necrotic response in the Best of All variety that was variably lethal, as compared to the faster inevitably lethal response induced by a prototype necrogenic D satellite variant in both tomato varieties. At higher temperatures (26.5 to 32 degrees C) an extremely fast-killing necrosis caused by CMV-Y itself was observed. The study demonstrates that in experiments on virus symptom modulation induced by CMV satellites, the nature of the helper virus, host plant varieties, as well as the environmental conditions should be precisely defined, and the effects of each parameter change determined separately.

Mosaic Viruses↗
PubMed1992

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts.

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.

Autoradiography↗
PubMed1992

Reduction of mortality rates of Bacillus thuringiensis var. israelensis aqueous suspensions due to freezing and thawing.

When studying the behavior (carry, dispersion, persistence) of Bacillus thuringiensis var. israelensis (B.t.i.) formulations used in the treatment of rivers or streams for black fly control, a large number of samples containing small quantities of B.t.i. are required for proper analysis. Freezing is a useful procedure to prevent enzymatic alteration or bacterial growth in samples before bioassays are to be performed. Using Aedes atropalpus neonate larvae, we studied the effect of freezing and thawing of B.t.i. aqueous suspensions by looking at mortality response parameters such as the slope and the LC50 of the probit regression. Initial concentration values of 1, 5, 10 and 20 mg/liter at the moment of freezing of the B.t.i. suspensions did not significantly affect toxicity. The number of freeze-thaw cycles greatly increased the LC50 values without much change to the slope of the log-probit regressions. We derived an equation that allowed us to compensate for the loss of toxicity of a given B.t.i. sample, knowing the number of freeze-thaw cycles.

Aedes↗
PubMed1990

Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus.

The two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of Mr 39,636 resembling in composition those of other large nepovirus satellite RNAs, a 5' leader sequence of 16 nucleotides and a 3' non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus.

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.

Base Sequence↗
PubMed1988

Cucumber mosaic virus-associated RNA 5. XI. Comparison of 14 CARNA 5 variants relates ability to induce tomato necrosis to a conserved nucleotide sequence.

Seven CARNA 5 (Cucumber mosaic virus-Associated RNA 5) variants were obtained from an Indonesian cucumber mosaic virus isolate (designated CMV-In) by serial passage of this virus in squash, Chenopodium quinoa, tobacco, and tomato. Bioassays of the isolated CARNA 5 variants for their capability to induce lethal necrosis in tomato allowed categorization of the CARNA 5 variants as either necrogenic or nonnecrogenic. Comparison of the CARNA 5 sequences with sequences previously published shows that in general CMV satellites display a high degree of nucleotide sequence conservation. Except for two sharply defined domains of variability, the sequences of the necrogenic CARNA 5s are essentially conserved, whereas those of the nonnecrogenic CARNA 5s possess at least nine additional hypervariable domains. In eight positions of the CARNA 5 sequences all necrogenic variants differ uniquely from those that are nonnecrogenic. All CMV satellites compared in this work possess open reading frames (ORFs), with equivalent initiation codons, capable of encoding polypeptides of significant lengths. Two equivalent or identical ORFs were found only in the necrogenic CARNA 5s, whereas a variable-length ORF was found in the sequences of all the CARNA 5 variants compared. These findings constitute a basis for experiments aimed at determining the biological significance, if any, of these ORFs.

Base Sequence↗
PubMed1983

Separation of the complementary strands of double-stranded cucumber mosaic virus-associated RNA 5 and peanut stunt virus-associated RNA 5.

The double-stranded forms of CARNA 5 and PARNA 5, viral satellites of the cucumovirus group, after denaturation and polyacrylamide gel electrophoresis under appropriate conditions allow their complementary strands to be separated and fractionated. These fractionated (+) and (-) strands are free of contaminating viral RNA fragments. They serve as templates for the preparation of cDNAs of opposite polarity, which are used to probe the viral RNA-dependent replication of CARNA 5 and PARNA 5.

Arachis↗
PubMedDate not supplied

Identification of a 334-ribonucleotide viral satellite as principal aetiological agent in a tomato necrosis epidemic.

Cucumber mosaic virus (CMV), a widespread and economically important virus of vegetable crops, often contains a satellite RNA, here designated CARNA-5 (for CMV-associated RNA 5). Viral satellites are small nucleic acids that are sequence-unrelated to, but replicatively dependent upon, the viral genome. They essentially are molecular parasites of their helper viruses, and thereby frequently modulate viral symptom expression. Some isolates of CARNA-5 change normally moderate CMV symptoms in tomato into a lethal disease named tomato necrosis; others ameliorate CMV symptoms in tomato and other important crop plants. Here we report on the identification and molecular characterization of a 334-nucleotide necrogenic CARNA-5 isolated from tomato fields in southern Italy, where a massive outbreak of lethal necrosis occurred in the summer of 1988. This is the first time that direct evidence is given for the involvement of a viral satellite in a crop disease of epidemic scale. The possible molecular interrelationships between plant, virus, satellite and other factors that influence the satellite-induced symptom modulation underlying such a catastrophe are discussed.

Fruit↗
PubMedDate not supplied

Cucumber-mosaic-virus-associated RNA-5. XII. Symptom-modulating effect is codetermined by the helper virus satellite replication support function.

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution endpoint bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found. The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.

Base Sequence↗