A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue

Tabler, Martin; Günther, Isolde; Kern, Rainer; Sänger, Heinz L.

doi:10.1016/0166-0934(89)90125-0

Cited by 15 publications

(12 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The viroid infection was done with longer-than-unit-length PSTVd (+) RNA of isolate KF-440-2, obtained by in vitro transcription (8). The infectivity assay was done by mechanical inoculation of 600ng viroid RNA with carborundum onto the stem of O. ramosa plants.…”

Section: Bioassay Methodologymentioning

confidence: 99%

Identification of Potato Spindle Tuber Viroid Small RNA inOrobanche Ramosaby Microarray

Ivanova

Vachev²,

Baev³

et al. 2010

Biotechnology & Biotechnological Equipment

View full text Add to dashboard Cite

Section: Bioassay Methodologymentioning

confidence: 99%

Identification of Potato Spindle Tuber Viroid Small RNA inOrobanche Ramosaby Microarray

Ivanova

Vachev²,

Baev³

et al. 2010

Biotechnology & Biotechnological Equipment

View full text Add to dashboard Cite

“…RNA was purified from PSTVd-infected and noninfected tomato leaves as described previously (51). Poly(A) ϩ mRNA purification was carried out with a Dynabead mRNA purification kit (Dynal, Oslo, Norway) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

A Bromodomain-Containing Protein from Tomato Specifically Binds Potato Spindle Tuber Viroid RNA In Vitro and In Vivo

Alba

Sägesser

Tabler

et al. 2003

J Virol

Self Cite

102

View full text Add to dashboard Cite

For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed.Viroids are the smallest known pathogens of higher plants. They consist of an infectious, single-stranded, covalently closed circular RNA molecule of ca. 250 to 400 nucleotides (nt). Viroids replicate autonomously, causing symptoms in specific host-viroid combinations of developmental disorders (9). Viroids are divided into two structural and functional distinct families. Potato spindle tuber viroid (PSTVd) is the type member of the Pospiviroidae and replicates in the nucleus. Avocado sunblotch viroid, the type member of the Avsunviroidae, replicates in the chloroplast of plant cells (14). Replication of both groups of viroids proceeds via RNA intermediates of complementary polarity. There is no evidence for viroid-encoded polypeptides. Thus, all genetic functions of the viroid replicon, such as cell entry and movement, replication, host specificity, and pathogenicity must be encoded in the RNA sequence and the resulting secondary structure, which is believed to provide specific signals for protein factors of the host. The predicted secondary structure of most viroids is a rod-like structure with five structural domains and an unusually high content of G and C bases (26). Despite the simple genome of viroids and the extensive knowledge of their structure and domains, little is known about host factors that interact with these structural elements.PSTVd replicates in the nucleus and accumulates eventually in the nucleolus as a circular single-stranded molecule, which is defined as positive polarity (44). Inhibition of PSTVd replication by alpha-amanitine indicates that viroid RNA strands of positive and negative polarity are most probably synthesized by the DNA-dependent RNA polymerase II (Pol II) (38,45). This enzyme can also accept PSTVd RNA as a template and "transcribe" it in vitro...

show abstract

“…The viroid to be sequenced is phenol-extracted together with the total nucleic acids from about 5-10g of leaf tissue 1-21] and purified by bidirectional polyacrylamide geMectrophoresis [19,21]. Since the minute amount of viroid RNA to be reversely transcribed…”

mentioning

confidence: 99%

“…Since the minute amount of viroid RNA to be reversely transcribed H. Puchta and H. L. S/inger cannot be detected by ethidium bromide staining, the location of the corresponding RNA band could only be determined by co-electrophoresis of a viroid marker in a separate lane. The gel slice containing the invisible traces of viroid RNA is excised according to the marker viroid and the nucleic acid of the viroid in question is eluted by isotachophoresis as previously described [21]. With the aid of M-MuLV reverse transcriptase and of an appropriate radioactively 5'-end-labeled DNA primer, the minute amount of viroid RNA is then transcribed into a viroid-specific ss cDNA fragment.…”

mentioning

confidence: 99%

“…The final products of the PCR were separated by PAGE under denaturing conditions on a 5% SVS gel [21] and visualized by autoradiography. 1 HSVDd cDNA amplified with the primer set pHSVd 39 and pCPFVd* 25 (see Table 1); 2 HSVd cDNA amplified with the primer set pHSVd 43 and pHCSVd* 24 (see Table 1); M Radioactively 5'-end-labeled Hinf I fragment of pBR 322 DNA used as size markers cDNAs.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR)

Puchta

Sänger

1989

Archives of Virology

View full text Add to dashboard Cite

The amount of viroid RNA required for sequence analysis can be reduced by five to six orders of magnitude when a modified "polymerase chain reaction" (PCR) is used for the amplification of the reversely transcribed, overlapping viroid cDNAs. By applying this procedure it is possible to establish the molecular structure also of those viroids which are present only in extremely low amounts in various crop plants and ornamentals.

show abstract

A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue

Cited by 15 publications

References 17 publications

Identification of Potato Spindle Tuber Viroid Small RNA inOrobanche Ramosaby Microarray

Identification of Potato Spindle Tuber Viroid Small RNA inOrobanche Ramosaby Microarray

A Bromodomain-Containing Protein from Tomato Specifically Binds Potato Spindle Tuber Viroid RNA In Vitro and In Vivo

Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR)

Contact Info

Product

Resources

About