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R H Symons

Publications and source records attributed to R H Symons.

At least 37 records · Page 2Linked to original sources
PubMed1994

Infectivity of linear monomeric transcripts of citrus exocortis viroid: terminal sequence requirements for processing.

Infectivity of linear monomeric viroid RNA transcripts is known to be dependent on the site of cloning of the viroid and the viroid and vector sequences at the termini of the RNA transcripts. We report here the further characterization of cloning site and sequence requirements using 11 different monomeric cDNA constructs of citrus exocortis viroid; these differed from one another by the site of cloning and by nonviroid residues adjacent to the 5' and 3' termini of the viroid sequence in the RNA transcript. The infectivity of these transcripts was assayed by inoculation onto tomato seedlings; all infectious transcripts were wild type, indicating accurate processing. The basic requirement for infectivity appeared to be the ability of the RNA transcripts to form a short double-stranded region of viroid and vector sequences at the junction of the two termini. The site of cloning of the viroid RNA used for cloning was not important as cDNA clones prepared at seven sites provided infectious transcripts.

Base Composition↗
PubMed1994

A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats.

An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura aurantiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill. x L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.

Base Sequence↗
PubMed1993

Is hammerhead self-cleavage involved in the replication of a virusoid in vivo?

The hammerhead self-cleavage reaction is generally considered to be involved in the in vivo production of (+) and (-) monomeric RNAs of the viroid-like, satellite RNA or virusoid of lucerne transient streak virus (vLTSV) from multimeric replicative intermediates, as part of the symmetrical rolling circle mechanism. To test this, three cDNA clones of vLTSV RNA, mutated at sites that inactivate in vitro self-cleavage of (-) RNA, were inoculated as excised plasmid cDNA inserts, together with helper virus LTSV, on Nicotiana clevelandii plants. As was predicted if hammerhead self-cleavage is involved in in vivo cleavage of (-) RNAs, high molecular weight (-) vLTSV RNAs were present in total RNA extracts from these mutant inoculated plants, but not in wild-type inoculated plants. Surprisingly however, the mutated virusoids also produced monomeric (-) RNAs in vivo. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed 8-20% of progeny contained reversions and pseudo-reversions of the introduced mutations. Hence, monomeric (-) RNAs were most likely produced by restoration of in vivo self-cleavage activity in the (-) RNA. Overall, the results indicate that mutations which disrupted self-cleavage in vitro also abolished the production of monomeric (-) RNAs in vivo and that hammerhead self-cleavage is involved in the cleavage of multimeric (-) RNAs to monomers. The observation that greater than 50% of the progeny cDNA clones generated from plants inoculated with mutant vLTSV contained base changes, compared to 20% from wild-type inoculated plants, may reflect an interesting adaptive response on the part of the mutated virusoids.

Amino Acid Sequence↗
PubMed1993

Isolates of citrus exocortis viroid recovered by host and tissue selection.

Isolates of citrus exocortis viroid (CEV) from a single sweet orange citrus source have been selected by sequential passage through the alternative hosts citron, Gynura aurantiaca, a hybrid tomato Lycopersicon esculentum x L. peruvianum, and from disorganized callus culture of the hybrid tomato. The distinctions in symptom expression, titre and electrophoretic mobility among the CEV isolates, operationally termed CEVc (citron), CEVg (Gynura), CEVt (tomato) and CEVcls (callus) are supported by characteristically different nucleotide sequences. The nucleotide sequence of full-length cDNA clones of CEVc purified from citron shows exchanges not reported for any previously described CEV variant. An unusual number of exchanges have been localized in the terminal domains of all the isolates analysed here. A common pattern of nucleotide exchanges, described as a 'tomato signature', can be detected in all of the isolates derived from hybrid tomato tissues.

Base Sequence↗
PubMed1991

Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs.

The plus and minus RNAs of the 247 nt avocado sunblotch viroid (ASBV) undergo site specific RNA self-cleavage reactions in vitro. As with several other self-cleaving RNAs, we proposed hammerhead secondary structures for the sequence around the site of self-cleavage of both RNAs. We have shown previously that, during transcription of a dimeric plus ASBV RNA, a double-hammerhead structure formed and was necessary for self-cleavage. Here, we show that the purified full-length dimeric plus RNA, when incubated under our standard self-cleavage conditions, also self-cleaved by a double-hammerhead structure. In contrast, a dimeric minus ASBV RNA self-cleaved by a double-hammerhead structure during transcription, but by a single-hammerhead structure after purification. This illustrates the importance of the pathway of folding of the RNA in determining which active self-cleaving structure is formed.

Base Sequence↗
PubMed1990

Structure, self-cleavage, and replication of two viroid-like satellite RNAs (virusoids) of subterranean clover mottle virus.

Both the genomic and viroid-like satellite RNAs (virusoids) from four subterranean clover mottle virus isolates described by Francki et al. were analyzed in detail. Restriction endonuclease mapping of cDNAs prepared from the genomic RNAs from all isolates showed that these RNAs are closely related if not identical. The two virusoids, which can occur together in the same isolate or individually, were sequenced and shown to be able to form highly base-paired viroid-like secondary structures. The left-hand portions of these structures are almost entirely homologous but the right-hand portions show little similarity. The plus, but not the minus, virusoid RNAs contain sequences that can form the hammerhead self-cleavage structure of certain other self-cleaving viroid, virusoid, and satellite RNAs. Plus, but not minus, RNA transcripts from cDNA clones self-cleaved essentially to completion at the predicted site during transcription in vitro. Northern blot analysis of infected leaf tissue extracts revealed the presence of an oligomeric series of plus RNAs (of monomer size and greater) but minus RNAs were present only as high molecular weight species of heterogeneous size. These findings are in agreement with the lack of minus RNA self-cleavage in vitro. Hence, these virusoid RNAs appear to replicate by a rolling-circle mechanism in which only the plus RNAs self-cleave to form monomeric RNAs.

Base Sequence↗
PubMed1989

Evolutionary relationship between luteoviruses and other RNA plant viruses based on sequence motifs in their putative RNA polymerases and nucleic acid helicases.

Comparative studies of sequence motifs in the RNA polymerases and nucleic acid helicases of positive-sense RNA plant viruses have provided a new scheme for the classification of these pathogens. We propose a new luteovirus supergroup which should be added to the already described Sindbisvirus-like and picornavirus-like supergroups. Sequence motifs of nucleic acid helicases and RNA polymerases which previously were considered to be specific for each of the two supergroups now occur together within this new supergroup. We propose that this new viral supergroup provides an evolutionary link between the other two supergroups.

Amino Acid Sequence↗
PubMed1989

RNA stem stability in the formation of a self-cleaving hammerhead structure.

The proposed single-hammerhead structure of the self-cleaving newt RNA is unstable due to a weak stem III and therefore is unable to mediate self-cleavage. A double-hammerhead structure with greater theoretical stability has been shown to mediate the self-cleavage of this RNA (Forster et al., 1988, Nature 334, 265). We have found that the double-hammerhead mediated self-cleavage reaction of a 40 base RNA containing the newt sequence (termed nCG) can be converted to a single-hammerhead reaction by increasing the size of stem III and/or of its loop, thereby enabling a single-hammerhead structure to form. In addition, the 5'-self-cleavage fragment of the nCG RNA can act in trans to mediate the self-cleavage of a full-length RNA by the formation of a partial double-hammerhead structure.

Base Sequence↗
PubMed1989

Mutagenesis analysis of a self-cleaving RNA.

The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage.

Base Sequence↗
PubMed1989

A catalytic 13-mer ribozyme.

A 13-mer oligoribonucleotide can act as a ribozyme for the specific self-cleavage of a 41-mer oligoribonucleotide substrate in the presence of Mg2+. The two sequences involved correspond to the self-cleavage hammerhead structure of the virusoid of lucerne transient streak virus. The Michaelis-menten kinetic parameters for the reaction were; Km 1.3 microM, Vmax 0.012 microM min-1, kcat 0.5 min-1. The 13-mer RNA is the smallest ribozyme so far reported. A DNA analogue of the 13-mer can not substitute for the RNA in the reaction.

Animals↗
PubMed1989

Comparative sequence studies of variants of avocado sunblotch viroid.

The nucleotide sequences of 16 variants of the 247 nucleotide avocado sunblotch viroid (ASBV), purified from leaves of three avocado trees in separate locations, have been determined and compared with that of the previously published ASBV SB-1 sequence (Symons, R. H., Nucl. Acids Res. 9, 6527-6537, 1981). Most of the nucleotide differences were found to occur in the left- and right-hand loops of the ASBV molecule. The number of residues in the sequence variants varied from 246 to 251. Two sequence variants contained nucleotide changes in the double hammerhead-like self-cleaving structure identified in ASBV RNA. RNA transcripts of dimeric cDNA clones of these sequence variants retained their in vitro self-cleavage activity.

Base Sequence↗
PubMed1989

Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts.

Avocado sunblotch viroid (ASBV), coconut cadang cadang viroid (CCCV), chrysanthemum stunt viroid (CSV) and potato spindle tuber viroid (PSTV) were detected in plant extracts by dot-blot hybridization using nonradioactive photobiotin-labelled nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were biotinylated with photobiotin and used as sonicated double-stranded DNA fragments. Using fresh leaf material, a general method (suitably modified for avocado tissue) was developed for the rapid preparation of purified nucleic acid extracts. Plant extracts from a range of field samples were spotted onto nitrocellulose, subjected to hybridization and the biotin-labelled DNA bound to the target nucleic acid was detected with an avidin-alkaline phosphatase conjugate. Under the stated hybridization and washing conditions, each individual viroid probe was specific. Each viroid was readily detected with a sensitivity similar to that obtained with the same (or a like) probe labelled with 32P. Healthy plant extracts gave colourless spots.

Affinity Labels↗
PubMed1989

Self-cleavage of RNA in the replication of small pathogens of plants and animals.

The ability of certain small, circular, pathogenic RNAs of plants and animals to self-cleave at specific sites in vitro in the complete absence of protein most likely plays a central role in their replication in vivo by a rolling circle mechanism. The self-cleavage of an RNA transcript from a satellite DNA of the newt indicates that this reaction is not limited to pathogenic RNAs. Further, the site-specific self-cleavage in trans by two separate RNA molecules suggests that such reactions may be important in gene regulation in normal cells as well as in the genesis of symptom expression on infection by RNA pathogens.

Animals↗