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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

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Published on: February 25, 2017

TYMV RNA replication in vivo: replicative intermediate is mainly single stranded.

M Garnier¹, R Mamoun, J M Bove

¹Laboratoire de Biologie Cellulaire et Moléculaire, INRA and Université de Bordeaux II, Domaine de la Grande Ferrade, 33140 Pont de la Maye, France.

|July 30, 1980

Summary

This summary is machine-generated.

This study developed a Pronase-RNase technique to detect double-stranded RNA (dsRNA) in plant virus-infected cells. Results indicate that dsRNA is an artifact of RNA extraction, not present in vivo.

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Area of Science:

Molecular Biology
Plant Virology
Biochemistry

Background:

Double-stranded RNA (dsRNA) is a key intermediate in the replication of many RNA viruses.
Accurate detection of dsRNA in situ is crucial for understanding viral replication strategies.

Purpose of the Study:

To develop and validate a Pronase-RNase technique for detecting dsRNA in glycol methacrylate-embedded ultrathin sections.
To investigate the in situ presence of TYMV-specific dsRNA in infected plant cells.

Main Methods:

Developed a Pronase-RNase digestion method for detecting dsRNA resistance to RNase at high ionic strength.
Utilized high-resolution autoradiography for dsRNA detection on ultrathin sections.
Compared in situ detection with RNA extraction and polyacrylamide gel electrophoresis.

Main Results:

The Pronase-RNase technique successfully detected dsRNA based on differential RNase sensitivity.
In vivo labeling of TYMV-infected leaves showed dsRNA as the main species after extraction.
However, in situ Pronase-RNase treatment revealed no dsRNA in glycol methacrylate-embedded sections, suggesting it's an artifact.

Conclusions:

Replicative intermediate RNA in TYMV-infected cells is predominantly single-stranded in vivo.
The observed dsRNA is likely an artifact generated during RNA extraction procedures.
This highlights the importance of in situ validation to avoid misinterpretation of molecular data.