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 ( 0.40 × 10) containing the 3a gene, and a short (Sh-BMV 3) fragment ( 0.28 × 10) 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.

Keyword(s): BMV and ribonuclease

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