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R H Symons

Publications and source records attributed to R H Symons.

At least 55 records · Page 3Linked to original sources
PubMed1988

Self-cleaving viroid and newt RNAs may only be active as dimers.

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.

Animals↗
PubMed1988

Coconut tinangaja viroid: sequence homology with coconut cadang-cadang viroid and other potato spindle tuber viroid related RNAs.

The nucleotide sequence of two variants of coconut tinangaja viroid (CTiV) were obtained. Both sequence variants are 254 nucleotide residues in size but differ in sequence at two positions. In comparisons with other viroids, the sequences and proposed secondary structure of CTiV show most homology with the 246 nucleotide residue variant of coconut cadang-cadang viroid (CCCV (246]. Several regions throughout the rod-like molecules of CTiV show significant structural and sequence homology with other RNAs related to potato spindle tuber viroid. These homologies include a centrally positioned uridine bulged G:C helix and a 17 nucleotide residue sequence found in common with hop stunt viroid in the left-hand end loop of both viroids.

Base Sequence↗
PubMed1988

Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3.

The nucleotide sequence of the RNA 3 of the Q-strain of cucumber mosaic virus (Q-CMV) has been reinvestigated and supporting partial amino acid sequence data obtained for the coat protein. Corrections to the previously published sequence of RNA 3 [A. R. Gould and R. H. Symons (1982) Eur. J. Biochem. 126, 217-226] result in changes to the size and composition of the putative 3a and coat proteins. Analysis of the nucleotide sequence revealed a 14-nucleotide sequence present in the intercistronic regions of the RNA 3 molecules of both Q-CMV and brome mosaic virus (BMV). This sequence, which is closely related to sequences previously detected in the 5'-untranslated region of Q-CMV and BMV RNAs 1 and 2 [M. A. Rezaian, R. H. V. Williams, and R. H. Symons (1985) Eur. J. Biochem. 150, 331-339], may be important in the control of RNA synthesis. Computer-assisted comparisons indicate an ancestral relationship between the 3a proteins of CMV, BMV, and alfalfa mosaic virus (AMV) and between the coat proteins of CMV and BMV. These comparisons significantly extend previous observations regarding the close evolutionary relationships within the plant tripartite virus group.

Amino Acid Sequence↗
PubMed1988

Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.

Alkaline Phosphatase↗
PubMed1987

Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens.

Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens.

Alkaline Phosphatase↗
PubMed1987

Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site.

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Recently, we showed that an in vitro-synthesized RNA containing 273 nucleotides of the 324-nucleotide virusoid of lucerne transient streak virus self-cleaves at a unique site. The reaction requires heating and snap cooling of the RNA and the subsequent addition of magnesium ions. Here, we test the 55-nucleotide, hammerhead-shaped, structural model proposed for the active site by preparing RNAs with both 5' and 3' terminal deletions. Results indicate that the hammerhead structure is sufficient and necessary for self-cleavage, that certain sequences prevent the formation of the hammerhead structure in the native virusoid RNA, and that an RNA molecule containing only 52 nucleotides is capable of an RNA-mediated reaction.

Base Sequence↗
PubMed1987

Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites.

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Virusoid replication appears to involve longer-than-unit-length plus and minus RNAs, indicating that unit-length plus RNA is generated by specific cleavage reactions. Here, we synthesize plus and minus partial-length RNAs of the 324-nucleotide virusoid from lucerne transient streak virus in vitro. Both RNAs self-cleave at a unique site in the presence of magnesium ions to give 5' hydroxyl and 2',3' cyclic phosphodiester termini. Conformations other than the native structures are necessary for cleavage. Similar secondary structures with considerable sequence homology are proposed for the active sites of these and other plant pathogenic RNAs. Our results are consistent with certain rolling-circle replication models.

Binding Sites↗
PubMed1987

Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus.

Photobiotin was used to prepare biotinylated, nonradioactive nucleic acid probes for the detection of the RNA of barley yellow dwarf virus (BYDV) in plant extracts. A 1.7-kb cDNA of the PAV isolate of BYDV in the plasmid pUC8 vector was biotinylated and then used intact or as sonicated double-stranded DNA fragments. Simple methods were developed for the preparation of partially purified nucleic acid extracts of cereals and their spotting, after formaldehyde treatment, onto nitrocellulose membranes. After hybridization, biotin-labelled DNA bound to BYDV RNA on the nitrocellulose was detected with an avidin-alkaline phosphatase conjugate. BYDV RNA was readily detected with a sensitivity similar to that found with the same probe labelled with 32P by nick translation. Healthy plant extracts gave colourless spots.

Affinity Labels↗
PubMed1987

Self-cleavage of RNA in the replication of viroids and virusoids.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Base Sequence↗
PubMed1986

Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid.

Self-cleavage of both plus and minus RNA transcripts of the 247-residue avocado sunblotch viroid (ASBV), prepared from tandem dimeric cDNA clones, occurs specifically at two sites in each transcript to give monomeric plus and minus species. The cleavage reaction occurs both during transcription and on incubation of purified transcripts at pH 8 and 37 degrees C in the presence of magnesium ions to give a 3'-terminal 2',3'-cyclic phosphate and a 5'-terminal hydroxyl group. Although the self-cleavage occurs at different sites in the ASBV molecule for the plus and minus species, very similar secondary structures with high sequence homology can be drawn at each site. The results are considered to provide further evidence that ASBV is replicated in vivo by a rolling circle mechanism involving non-enzymic cleavage of high molecular weight RNA precursors of ASBV.

Genes, Viral↗
PubMed1986

Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene.

The interaction in vitro of the RNA of the Q-strain of cucumber mosaic virus (CMV) with its satellite RNA (sat-RNA) has been studied. In hybridisation reactions containing 30% formamide at 45 degrees, sat-RNA binds to CMV RNA 3 and 4 but not to CMV RNA 1 and 2 or RNA from tobacco mosaic virus and alfalfa mosaic virus. The viral coat protein gene present in RNA 3 and 4 contains the site of binding but this region does not contain complementary sequences of any significant length to the sat-RNA sequence. However, the optimum alignment of short complementary sequences present in these regions revealed a stable structure in which it is proposed that sat-RNA twists around the coat protein gene so that two separate blocks of nucleotides in sat-RNA base pair in opposite directions with two adjacent blocks in the coat protein gene to form a knot-like structure. The binding site is a region of 33 nucleotides within the coding region of the coat protein gene which base pairs with residues 98-113 and 134-152 of sat-RNA. The possibility of the binding region of sat-RNA functioning as an "anti-sense" sequence in regulation of the viral coat protein synthesis is discussed.

Base Sequence↗
PubMed1985

Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors.

Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5'GGATCCCCGGG 3', in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5'-end of the CEV insert to 5' GGATCCCC(T,A)GG 3' gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.

Base Sequence↗
PubMed1985

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs.

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.

Amino Acid Sequence↗
PubMed1985

Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity.

Full-length double-stranded cDNA was prepared from purified circular RNA of two new Australian field isolates of citrus exocortis viroid (CEV) using two synthetic oligodeoxynucleotide primers. The cDNA was then cloned into the phage vector M13mp9 for sequence analysis. Sequencing of nine cDNA clones of isolate CEV-DE30 and eleven cDNA clones of isolate CEV-J indicated that both isolates consisted of a mixture of viroid species and led to the discovery of eleven new sequence variants of CEV. These new variants, together with the six reported previously, form two classes of sequence which differ by a minimum of 26 nucleotides in a total of 370 to 375 residues. These two classes correlate with two biologically distinct groups when propagated on tomato plants where one produces severe symptoms and the other gives rise to mild symptoms. Two regions of the native structure of CEV, comprising 18% of the total residues, differ between the sequence variants of mild and severe isolates. Whether or not both of these regions are essential for the variation in pathogenicity has yet to be determined.

Base Sequence↗
PubMed1985

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.

Azides↗
PubMed1985

Dot-blot procedure with [32P]DNA probes for the sensitive detection of avocado sunblotch and other viroids in plants.

Avocado sunblotch viroid (ASBV) has been detected down to a level of about 20 pg per gram fresh weight of leaves by the use of a dot-blot hybridization procedure and partially purified nucleic acid extracts. Three [32P]DNA probes were compared, two prepared from full-length ASBV clones in the single-strand M13mp93 vector and the other by primer extension on purified ASBV. All three probes gave the same sensitivity of detection of ASBV. The methods developed have also been used successfully for the routine detection of potato spindle tuber viroid, citrus exocortis viroid, and coconut cadang cadang viroid.

Cloning, Molecular↗
PubMed1985

Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution.

On the basis of sequence homology a model is proposed for five structural and functional domains in viroids. These domains include (i) a conserved central region capable of forming two alternative structures that may regulate two phases of the viroid replication cycle, (ii) a region associated with pathogenicity, (iii) a domain with high sequence variability, (iv and v) two terminal domains that are interchangeable between viroids. That the evolution of viroids has involved RNA rearrangements of domains is supported by the partial duplication of coconut cadang cadang viroid, which arises de novo during each infection. Similar RNA rearrangements have been established for animal viral defective interfering RNAs, which arise by some form of discontinuous transcription. This mechanism could account for the origin of viroids and also RNA viruses, whereby modules of genetic information may have undergone repeated exchange between RNA pathogens and the RNA of their hosts.

Base Sequence↗