RNase-resistance

RNase protection assays (RPA) are based on the property of RNase to digest ii RNA, but not ds RNA, and its principles and applications resemble those of SI analysis (Lynn et al., 1983 Zinn et at, 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUC118, pT7, etc.. Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the... 

Heterogeneous RNA contains various regions resistant to RNase treatment one is the poly A sequence attached to the tail (see below), the others are various base sequences. The amount of RNase-resistant sequence varies considerably from species to species, and such portions of the hnRNA are referred to as... 

The two known functions of the viral coat proteins, protection against RNase and specific adsorption to cells, are affected by DEP to a small (RNase resistance) or large (adsorbability) extent and in the latter case can be restored completely by addition of DEAE-dextran. But nevertheless, the infectivity of DEP-treated poliovirus is the same as that of isolated viral RNA. This shows clearly that loss of RNase resistance is not responsible for the low infectivity of the isolated viral RNA. It also appears very unlikely that the difference in adsorbability between intact virus and isolated RNA and DEP-inactivated virus is responsible for the low infectivity of the latter. However, it is possible that native virus particles enter the cell at specific membrane loci. Provided that neither loss of RNase resistance nor adsorbability are responsible for the comparatively low infectivity of naked RNA and of virus particles inactivated by heat or DEP, the viral coat proteins must have yet another function, so far unidentified. 

Most of the input counts band at the position close to RF-RNA, whereas the majority of the newly incorporated counts band between RF-RNA and single-stranded RNA, closer to the single-stranded RNA. Different RNA fractions (I, II, III) from the gradient were pooled and their RNase resistance... 

Up to 8% of the newly synthesized RNA present in single-stranded form anneals to poUovirus plus strand and up to 9% of the added poliovirus plus-strand RNA is protected from RNase digestion by the newly synthesized RNA. The conversion of both viral RNA and newly synthesized RNA from an RNase-sensitive to an RNase-resistant RNA is dependent on the relative proportions of the RNA. 

These results indicate that the poliovirus RF-RNA served as a template for the synthesis of RNA complementary to viral RNA (i.e. minus-strand RNA). Separated poliovirus minus- and plus-strand RNAs anneal with an efficiency of 60 % under our experimental conditions (RNA concentrations of 5 (xg/ml, assay of RNase resistance with Tj RNase and RNase I). This value is considerably lower than that found with reovirus (Shatkin and Banerjee, 1970) or phage RF-RNA (Bishop and Levintow, 1971). Part of the reannealed poliovirus RNA is found in a structure resembhng RI-RNA (Bishop and Levintow, 1971) (Koch and Vollertsen, unpubhshed). Therefore, the estimate of the synthesis of pohovirus-specific RNA in E. coli determined by RNase resistance of the annealed RNA represents only a minimal... 

In other experiments, poliovirus minus-strand RNA was used to detect newly synthesized viral plus-strand RNA. The extent of conversion of newly synthesized RNA from RNase-sensitive to RNase-resistant form was again found to depend on the relative proportions of the two RNAs. However, the... 

In electron microscopic studies Verhey, Moyer, and Iverson (1965) have demonstrated that microsomes of unfertilized sea urchin eggs differ from those of fertilized eggs in being associated with RNase-resistant, trypsin-labile material. 

Kaulenas and Fairbaim (1966) have presented evidence for the presence of RNase-insensitive, inactive, ribosomal aggregates in precleavage eggs of Ascaris lumbricoides. Trypsin treatment of the microsomal pellet sensitizes the aggregates to dissociation by RNase and, at the same time, activates them for protein synthesis. The proportion of RNase-resistant, inactive polysomes decreases with development. Simi-... 

---