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D Rochon

Publications and source records attributed to D Rochon.

18 recordsLinked to original sources
PubMed2003

Molecular analysis of the cucumber leaf spot virus genome.

Full-length clones of the genome of the Aureusvirus, Cucumber leaf spot virus (CLSV), have been constructed and infectious T7 polymerase derived synthetic transcripts have been produced. Mutational analysis of the genome indicates a role for p84 in viral RNA replication, the CP in systemic movement, p27 in viral cell-to-cell movement and p17 in symptom induction. A CLSV mutant lacking ORFs for the CP, p27 and p17 (CLSV YX) was capable of replication and systemic movement in transgenic Nicotiana benthamiana plants expressing the Red clover necrotic mosaic virus (RCNMV) movement protein (MP) suggesting that p25 and p84 are sufficient for viral RNA replication and that the RCNMV MP can permit CLSV cell-to-cell as well as systemic movement. Moreover, CLSV YX induced severe necrosis in both inoculated and uninoculated leaves of transgenic plants suggesting that CLSV p25 and/or p84 are important determinants of the necrotic phenotype. Another mutant similar to CLSV YX but expressing only limited amino-terminal portions of CP, p27 and p17 failed to produce necrosis or to move systemically in RCNMV MP transgenic N. benthamiana plants. These results suggest that these short translated regions or cis-acting sequences present in the CLSV CP, p27 and/or p17 ORFs suppress the necrosis induced by p25/p84 and also suppress systemic movement mediated by the RCNMV MP.

Amino Acid Sequence↗
PubMed2001

Synapse-glia interactions at the mammalian neuromuscular junction.

Perisynaptic Schwann cells (PSCs) play critical roles in regulating and stabilizing nerve terminals at the mammalian neuromuscular junction (NMJ). However, although these functions are likely regulated by the synaptic properties, the interactions of PSCs with the synaptic elements are not known. Therefore, our goal was to study the interactions between mammalian PSCs in situ and the presynaptic terminals using changes in intracellular Ca(2+) as an indicator of cell activity. Motor nerve stimulation induced an increase in intracellular Ca(2+) in PSCs, and this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of acetylcholine induced Ca(2+) responses that were blocked by the muscarinic antagonist atropine and mimicked by the muscarinic agonist muscarine. The nicotinic antagonist alpha-bungarotoxin had no effect on Ca(2+) responses induced by acetylcholine. Local application of the cotransmitter ATP induced Ca(2+) responses that were unaffected by the P2 antagonist suramin, whereas local application of adenosine induced Ca(2+) responses that were greatly reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). However, the presence of the A1 antagonist in the perfusate did not block responses induced by ATP. Ca(2+) responses evoked by stimulation of the motor nerve were reduced in the presence of CPT, whereas atropine almost completely abolished them. Ca(2+) responses were further reduced when both antagonists were present simultaneously. Hence, PSCs at the mammalian NMJ respond to the release of neurotransmitter induced by stimulation of the motor nerve through the activation of muscarinic and adenosine A1 receptors.

Acetylcholine↗
PubMed2001

Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment.

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138-146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.

Amino Acid Substitution↗
PubMed2000

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

Animals↗
PubMed1999

The ion coupling and organic substrate specificities of osmoregulatory transporter ProP in Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily, mediates osmoprotective proline or glycine betaine accumulation by bacteria exposed to high osmolality environments. Morpholinopropane sulfonic acid, a common constituent of microbiological media, accumulates in osmoadapting E. coli cells but it is not osmoprotective and it did not influence proP transcription or ProP activity. The apparent K(m) for proline uptake via ProP increased with decreasing pH in the range 7.5-4. ProP-dependent proline uptake by de-energized bacteria was associated with alkalinization of the external medium. Thus ProP mediates cotransport of H(+) and zwitterionic proline and a transporter functional group with a pK(a) of 5-6 is implicated in catalysis. Exogenous proline or glycine betaine elicits K(+) release from osmoadapting E. coli cells and ProP activity is stimulated by exogenous K(+). However, uptake of proline or glycine betaine stimulated K(+) efflux from K(+)-loaded bacteria which expressed either ProP or alternative, osmoregulatory transporter ProU. This indicated that ProP was unlikely to mediate K(+) efflux. Zwitterions ectoine, pipecolate, proline betaine, N,N-dimethylglycine, carnitine and 1-carboxymethylpyridinium were identified as alternative ProP substrates. Choline, a cation and a structural analogue of glycine betaine, was a low affinity inhibitor but not a substrate of ProP.

Bacterial Proteins↗
PubMed1999

Both RNA rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants.

The putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (CLSV) was inserted into an infectious cucumber necrosis tombusvirus (CNV) cDNA clone containing a deletion in the cell-to-cell movement protein gene. Approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric CNV/CLSV cDNA clones developed systemic symptoms 7-19 days postinoculation. Reverse transcription-polymerase chain reaction and sequence analysis of virus obtained from systemically infected leaves indicated that both point mutation and RNA rearrangement (deletion) contributed to the formation of movement competent CNV/CLSV hybrid viruses. The hybrid viruses were found to accumulate to high levels in infected plants, to form stable virions, and to be mechanically transmissible. In addition, a hybrid virus that lacked 50 amino acids at the carboxyl-terminal region of CLSV p27 was still capable of facilitating CNV movement. These data provide experimental evidence for the role of CLSV p27 in viral cell-to-cell movement and demonstrate that p27 can enable efficient movement of the CNV genome. Moreover, the data show that RNA rearrangements known to occur during CNV RNA replication can contribute to rapid evolution of the CNV genome.

Amino Acid Sequence↗
PubMed1998

Phat phacts.

Explore the source record for details and available documents.

Body Composition↗
PubMed1995

Mutational analysis of the cucumber necrosis virus coat protein gene.

A series of frameshift, deletion, and inversion mutations were made in the coat protein (CP) gene of the icosahedral cucumber necrosis tombusvirus (CNV) to investigate the role of the CP protruding (P) domain in the production of virus particles and, also, to investigate the basis for the accumulation of CP deletion derivatives previously reported in plants inoculated with PD(-), a P-domainless CNV CP mutant. P-domainless coat protein subunit could be detected in extracts of CP mutant-infected plants; however, virus-like particles could not, suggesting that the P domain is essential for tombusvirus particle assembly and/or stability. In addition, each of the P-domain mutants analyzed invariably produced coat protein deletion derivatives in infected plants whereas shell domain mutants rarely produced deletion derivatives. Finally, P-domain inversion and deletion mutants accumulated deletion derivatives very rapidly in comparison to P-domain frameshift mutants. Protoplast studies show that PD(-) RNA inoculum does not undergo further deletion in infected protoplasts, suggesting that PD(-) CP deletion derivatives preferentially accumulate in plants because they have a greater capacity for cell-to-cell movement.

Base Sequence↗
PubMed1995

Nucleotide sequence of tomato ringspot virus RNA1.

The nucleotide sequence of tomato ringspot nepovirus (TomRSV) RNA1 has been determined. TomRSV RNA1 is 8214 nucleotides in length, excluding the 3' poly(A) tail, and contains a single long open reading frame (ORF) of 6591 nucleotides beginning at the first AUG codon at nucleotide position 78. This ORF accounts for 80% of the RNA1 sequence and would give rise to a polyprotein with a predicted molecular mass of 244 kDa. Amino acid sequence comparisons between portions of the TomRSV RNA1-encoded polyprotein and proteins encoded by several members of the picornavirus superfamily have provided information concerning the genomic organization and putative functions of TomRSV-encoded proteins. The putative TomRSV protease retains a conserved histidine residue present in the proteases encoded by members of the como-, poty- and poliovirus groups which is thought to be involved in dipeptide cleavage site recognition. Interestingly, this histidine residue is replaced by a leucine in the proteases of other sequenced nepoviruses. This suggests that the TomRSV protease shares dipeptide cleavage site specificity with that of como-, poty- and picornaviruses rather than the other nepoviruses.

Amino Acid Sequence↗
PubMed1989

Organization of tomato bushy stunt virus genome: characterization of the coat protein gene and the 3' terminus.

We have synthesized cDNA clones of the genome of the cherry strain of tomato bushy stunt virus (TBSV-cherry) and have used them as hybridization probes to identify and position two 3' coterminal subgenomic RNAs of approximately 2.2 and 0.9 kilobases (kb) in length. The 5' termini of the two subgenomic RNAs have been mapped to positions located 2156 and 936 nucleotides respectively from the 3' terminus of the viral genome. The nucleotide sequence of cDNA clones encompassing the region of the genome containing both of the subgenomic RNAs has been determined. The sequence data indicate that two nested open reading frames (ORFs) occur in the most 3' proximal location on the genome suggesting that the 0.9-kb subgenomic RNA potentially encodes two polypeptides of 19,397 and 21,610 Da. Comparison of the amino acid sequence of a potential translation product of 41,024 Da encoded by the first ORF of the 2.2-kb subgenomic RNA with the published capsid protein amino acid sequence of the BS-3 strain of TBSV indicates that the 2.2-kb subgenomic RNA encodes the capsid protein. The TBSV coat protein cistron is located internally on the genome and thus its genetic organization differs from that reported for most other small, spherical viruses with monopartite genomes. Amino acid sequence comparisons of analogous regions of the cucumber necrosis virus (CNV) genome confirms a close relationship between the viruses.

Amino Acid Sequence↗
PubMed1988

Characterization of the TMV encapsidation initiation site on 18S rRNA.

Tobacco Mosaic Virus capsid protein oligomers react with and encapsidate 18S rRNA from both plant and mammalian sources in vitro. The site (ei) in 18S rRNA which reacts with capsid protein to initiate the packaging reaction has been localized and partially characterized by testing the ability of transcripts from different regions of a cloned Cucurbita pepo rDNA repeat unit to become encapsidated. The 18S rRNA ei is found to react more slowly with capsid protein than does the functional virion ei and to lie within a 43 nucleotide region which starts at position 157 from the 5' terminus of 18S rRNA. When 6 nucleotides are removed from the 5' end, the remaining 37 nucleotide segment is still reactive, but with reduced efficiency. The primary structure of the reactive segment has limited similarity to the virion ei and can be folded into a stem-loop. The first 18 nucleotides of the ei region is highly conserved from an evolutionary standpoint and this may account for the ability of 18S rRNAs from both plant and mammalian sources to be encapsidated.

Capsid↗
PubMed1986

Encapsidation of 18 S rRNA by tobacco mosaic virus coat protein.

It has been reported that tobacco mosaic virus capsid protein encapsidates discretesized truncated portions of host 18 S rRNA in vitro. This paper presents further information concerning the nature and specificity of this reaction. We have found that it is only the 5' portions of 18 S rRNA that are encapsidated. The structure recognized by capsid protein is highly conserved; bovine as well as plant 18 S rRNA becomes encapsidated. It is further demonstrated that assembly of 18 S rRNA is slow in comparison to assembly of TMV RNA and that this is due to a slow rate of initiation. Synthetic 18 S rRNA, prepared by in vitro transcription of an 18 S rRNA coding sequence, differs from native 18 S rRNA in that full length, rather than a truncated portion, is encapsidated. The possible reasons for this are discussed.

Base Sequence↗