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Deletion of the Intercistronic Poly(A) Tract from Brome Mosaic Virus RNA 3 by Ribonuclease H and Its Restoration in Progeny of the Religated RNA 3

Department of Virology and A.N. Belozersky Laboratory of Moscow State University, Moscow 119899, U.S.S.R.

The dicistronic genomic RNA 3 of brome mosaic virus (BMV) was used in experiments on site-specific cleavage by RNase H and subsequent religation of large BMV RNA 3 fragments with T4 RNA ligase. BMV RNA 3 was cleaved at the intercistronic poly(A) tract into two fragments: a long (L-BMV 3) 5'-terminal fragment (M_r 0.40 x 10⁶) containing the 3a gene, and a short (Sh-BMV 3) fragment (M_r 0.28 x 10⁶) containing the coat protein gene and the 3'-terminal tRNA-like tyrosine-accepting structure. Two or three adenylate residues were present at the 5' end of Sh-BMV 3 and one adenylate at the 3' end of L-BMV 3. After religation of these RNA fragments BMV RNA 3 was constructed with a deletion including the entire intercistronic poly(A) tract but not the flanking sequences. The religated RNA 3 replicated in wheat plants co-inoculated with BMV RNA 1 and RNA 2. The normal poly(A) tract was restored in progeny BMV RNA 3 during the course of replication.

Keywords: BMV, ribonuclease

Received 5 December 1988; accepted 10 May 1989.