Potato Spindle Tuber Disease: Procedures for the Detection of Viroid RN'A and Certification of Disease-Free Potato Tubers

Morris, T. Jack; Smith, E. M.

doi:10.1094/phyto-67-145

Cited by 92 publications

(47 citation statements)

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“…Two-dimensional gel electrophoretic analysis (6,7) was carried out as described (8) by using a 5% nondenaturing gel containing a Tris acetate buffer (0.040 M Tris, pH 7.2/0.020 M sodium acetate/0.001 M EDTA; ref. 6) for the first dimension and a 7 M urea gel (9) containing a Tris borate buffer (0.045 M Tris, pH 8.2/0.044 M boric acid/0.0014 M EDTA) for the second dimension.…”

Section: Methodsmentioning

confidence: 99%

“…Oligonucleotides were recovered either by soaking excised gel bands overnight at 37°C in 100 ,ul of water or by eluting fingerprint spots from DEAE thin-layer plates (13). Secondary enzymatic digestion of oligonucleotides and fractionation of the products were carried out as described (11,13,15 25 ,ug of nucleic acids from the LiCl supernatant fraction purified from PSTV-infected tomato plants as described (6,16,17) was dissolved in Tris acetate buffer (6) and then exposed to UV light for 7 min. In the two-dimensional gel pattern of nucleic acids from a control sample (A), an arrow identifies the position of circular PSTV.…”

Section: Methodsmentioning

confidence: 99%

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Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

Branch¹,

Benenfeld²,

Robertson³

1985

Proc. Natl. Acad. Sci. U.S.A.

106

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The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA frgerprinting and secondary analysis (and additional primer extension studies in the case of the viroid). Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (0) both crosslinks occur in an identical eight-base context, with the sequence 5' GGGAA 3' on one side and the sequence 5' UAC 3' on the other; (it) both crosslinks connect bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as singlestranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules. MATERIALS AND METHODSGel Procedures. Two-dimensional gel electrophoretic analysis (6, 7) was carried out as described (8) (Sankyo, Calbiochem) or pancreatic RNase (Worthington) at 1 mg/ml in the presence of 10 ,g of E. coli tRNA under conditions as described (11,13,14). After digestion, samples were either fractionated by electrophoresis in 20% polyacrylamide gels containing 7 M urea for one-dimensional analysis or were spotted onto cellulose acetate strips as the first step toward the preparation of standard two-dimensional fingerprints (13).Classical Secondary Analysis. Oligonucleotides were recovered either by soaking excised gel bands overnight at 37°C in 100 ,ul of water or by eluting fingerprint spots from DEAE thin-layer plates (13). Secondary enzymatic digestion of oligonucleotides and fractionation of the products were carried out as described (11,13,15 6590The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

Branch¹,

Benenfeld²,

Robertson³

1985

Proc. Natl. Acad. Sci. U.S.A.

106

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“…Low molecular weight RNA was prepared from 6 g (fresh weight) of individual uninfected, PSTV-infected, and CEV-infected tomato plants by the extraction procedure of Morris and Smith (26 (27), and concentrated by CF11 cellulose chromatography (28) and ethanol precipitation. When the total amount of RNA present in the gel band was in the range of 0.05-1.0 ,ug, this procedure was able to produce intact RNA suitable for subsequent in vitro iodination to specific activities in the range of 40-160 X 106 dpm/,ug.…”

Section: Methodsmentioning

confidence: 99%

Potato spindle tuber and citrus exocortis viroids undergo no major sequence changes during replication in two different hosts

Dickson¹,

Diener²,

Robertson³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Potato spindle tuber viroid and citrus exocortis viroid, each purified from tomato (Lycopersicon esculentum) and from Gynura aurantiaca, were iodinated in vitro with 125I, digested with ribonuclease T1, and subjected to two-dimensional RNA fingerprinting analysis. With the exception of minor variations, each viroid retained its distinctive fingerprint pattern irrespective of the host species from which it was isolated. We conclude that the nucleotide sequences of these viroids are principally determined by the infecting viroid and not by the host.Viroids are subviral plant pathogens composed exclusively of low molecular weight RNA (1) of low complexity (2-4) that cause a number of severe disease symptoms (usually including stunted growth) in a variety of plants (5). Viroids have singlestranded genomes with highly developed secondary structures (6). Various proportions of the infectious RNA (20%-99%) may be isolated as covalently closed circles (7-9). Viroids have genomes about one-tenth the size of the smallest known bacteriophage genomes (10) (about 350 nucleotides; ref. 9). Viroids appear to exist without the protection of a capsid (11,12) and lack detectable messenger RNA activity (13,14). Demonstration that viroid replication occurs in host nuclei (15) The observation that potato spindle tuber viroid (PSTV) and citrus exocortis viroid (CEV) produce similar disease symptoms across a common host range led to suggestions that they might be independent isolates of the same pathogen (20)(21)(22) 125I (4, 24). This approach has been used in the studies reported here to determine whether the genomes of PSTV and CEV undergo major sequence alterations after replication in tomato and Gynura plants. Polyacrylamide Gel Electrophoresis. All RNA species were subjected to a final fractionation on eight-slot slab gels 20 X 20 X 0.3 cm containing 5% acrylamide (Canalco) and 0.25% bisacrylamide (Canalco). Gels were made up and samples were electrophoresed in 40 mM Tris-HCI/20 mM sodium acetate/i mM EDTA at pH 7.2 (26). After 5 min of electrophoresis, samples were electrophoresed at 4°at 160 V (75 mA) for 5 hr, by which time the bromphenol blue marker had migrated 10 cm. The gels were then stained for 15 min in 20 ,ug of ethidium bromide per ml/1 mM EDTA, destained in 1 mM EDTA for an additional 15 min, and photographed. Gel bands of interest were excised and stored at -700 until extraction; the gels were rephotographed to record accuracy of excision. Extraction of RNA from Gel Bands. RNA was extracted by homogenization of the gel bands in phenol and 50 mM TrisHCl/0.1 M sodium chloride/i mM EDTA at pH 7.0, freed of acrylamide by hydroxylapatite chromatography (27), and concentrated by CF11 cellulose chromatography (28) and ethanol precipitation. When the total amount of RNA present in the gel band was in the range of 0.05-1.0 ,ug, this procedure was able to produce intact RNA suitable for subsequent in vitro iodination to specific activities in the range of 40-160 X 106 dpm/,ug.In Vitro Iodination of RNA. Commerford...

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“…is another method for PSTV detection (Morris and Smith 1977;Singh 1982) (Singh and Crowley 1985). A recent modification of the PAGE method, returnpolyacrylamide gel electrophoresis (R-PAGE) (Schumacher et al 1986) can increase sensitivity (Singh and Boucher 1987) Nucleic acids were extracted as previously described (Singh 1984) For personal use only.…”

Section: Polyacrylamide Gel Electrophoresis (Page)mentioning

confidence: 99%

A Survey of the Incidence of Potato Spindle Tuber Viroid in Prince Edward Island Using Two Testing Methods

Singh¹,

DeHaan²,

Jaswal³

1988

Can. J. Plant Sci.

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Potato Spindle Tuber Disease: Procedures for the Detection of Viroid RN'A and Certification of Disease-Free Potato Tubers

Cited by 92 publications

References 0 publications

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

Potato spindle tuber and citrus exocortis viroids undergo no major sequence changes during replication in two different hosts

A Survey of the Incidence of Potato Spindle Tuber Viroid in Prince Edward Island Using Two Testing Methods

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