Site‐specific enzymatic cleavage of TMV RNA directed by deoxyribo‐ and chimeric (deoxyribo‐ribo)oligonucleotides

Abstract

The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo-ribo)nucleotide with an internucleotide pyrophosphate bond Site specificity; RNA cleavage; RNase H; Oligo(deoxyribo-ribo)nucleotide

Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro

“…After 20-fold dilution with 5 mM-MgClz, complexes were adsorbed onto carboncoated rhodium-plated copper grids, negatively stained with 1 ~ (w/v) uranyl acetate and viewed in a Jeol 1200EX electron microscope. The lengths of nucleocapsids were measured directly in 200 of these complexes using the internal scale-bars, checked against virus particle diameter (18nm Uncoated RNA leader in TMV 771 cDNA to unencapsidated TMV RNA or to buffer-washed TMV particles, and subsequent digestion with RNase H (Pharmacia), were performed as described by Minshull & Hunt (1986) or Atabekov et al (1988). Each lyophilized eDNA was dissolved in water to a concentration of 50 to 450 IXM and added to the appropriate TMV template at cDNA :genome ratios between 100 : 1 and 1000 : 1.…”

“…RNase H Six short DNAs (cDNA1 to 6) complementary to portions of the 5'-proximal sequence of TMV RNA (residues 1 to 117), and one to the extreme 3' end of TMV RNA (cDNA7), were used to target RNA digestion by RNase H (Minshull & Hunt, 1986;Atabekov et al, 1988) prior to incubation of virions or unencapsidated TMV RNA in RRL. Sequence details of the cDNAs, the genome coordinates of their target sequence(s) and the standard cDNA : TMV genome ratios used are given in Table 1.…”

“…Their quantitative effects (with or without RNase H treatment) on RRL translation of pH 7-2-, 8-2-or 9.2-treated TMV particles or unencapsidated TMV RNA are summarized in Table 2. cDNA : TMV genome ratios varied between 100 : 1 and 1000 : 1 and, in some experiments, annealing was performed at 60 °C (Atabekov et al, 1988) rather than at 30 °C (Minshull & Hunt, 1986). Qualitative effects on the polypeptides encoded by unencapsidated TMV RNA or pH 7-2-washed virions are shown in Fig.…”

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

“…The method of site-specific cleavage with RNase H has been applied to different positions in diverse high Mr RNAs (Stepanova et al, 1979;Donis-Keller, 1979;Metelev et al, 1980;Dolja et al, 1982;Rodionova et al, 1983;Atabekov et al, 1988). In the case of BSMV RNAs and BMV RNA 3 the cleavage was performed at internal poly(A) tracts at different positions in the two viral RNAs.…”

“…The internal poly(A) tract could be restored in progeny RNA in the course of RNA 3 replication. Some aspects of this work have been reported in a preliminary communication (Tyulkina et al, 1988).…”