TYMV RNA replication in vivo: replicative intermediate is mainly single stranded.
We have used an in vitro labeled, membrane-bound ds[3H]RNA (C. Mouches, C. Bove, and J. M. Bove, 1974, Virology 58, 409-423) to develop a technique by which dsRNA can be detected on glycol methacrylate-embedded ultrathin sections by its resistance to RNase digestion at high but not at low ionic strength. The RNase-resistant ds[3H]RNA is detected on the sections by high-resolution autoradiography. The technique requires that prior to RNase digestion the ultrathin sections be first submitted to a Pronase step. This is accomplished by floating the sections on Pronase (1 mg/ml) for 15 min at 30 degrees , washing the sections three times, and digesting with RNase at high ionic strength for 90 min at 30 degrees . When the washing steps and the RNase hydrolysis are carried out at low ionic strength, dsRNA is completely hydrolyzed. We have applied the Pronase-RNase technique to investigate whether TYMV-specific dsRNA exists in situ after in vivo labeling of the RNAs. [3H]Uridine was fed for 1, 3, and 6 hr through the petiole of healthy or TYMV-infected leaves in the presence or absence of actinomycin D. For a given treatment aliquots of the same leaves were used for three parallel experiments: (1) Araldite embedding for autoradiography; (2) glycol methacrylate embedding for enzyme digestion and autoradiography; and (3) RNA extraction and analysis by polyacrylamide gel electrophoresis. These experiments have shown that after a [3H]uridine absorption by TYMV-infected leaves for 1 hr, in the presence of actinomycin D:dsRNA was the main labeled RNA species seen on the gels; Araldite-embedded sections were labeled specifically at the chloroplast periphery, while glycol methacrylate-embedded sections, submitted to the Pronase-RNase technique to detect dsRNA, were found to contain no dsRNA. Thus replicative intermediate RNA in TYMV-infected cells is essentially single stranded in vivo, and dsRNA is an artifact generated during the RNA extraction procedure.
Garnier
M
M
Laboratoire de Biologie Cellulaire et Moléculaire, INRA and Université de Bordeaux II, Domaine de la Grande Ferrade, 33140 Pont de la Maye, France.
Mamoun
R
R
Bove
J M
JM