Structure of RNA Tumor Virus Genome

Part I of the thesis describes the characterization of the RNA structure of the RNA tumor viruses. The genome of type C RNA tumor viruses is an RNA complex which sediments at "50-60"S in a nondenaturing aqueous electrolyte. Upon exposure to denaturing conditions, it can be dissociated into subunits of "30-40"S plus small "4-10"S RNA's. The structure and molecular weight of the "50-60"S and "30-40"S RNA species have been studied by electronmicroscopy, gel electrophoresis and sedimentation analysis. It was found that type C viruses, isolated from different origins (feline, murine, simian and baboon) all contain as their genomes "50-60"S RNA species which have similar secondary structure patterns. The "50-60"S molecule has a molecular length about 16-20 Kb (~6x106 daltons) as measured by electronmicroscopy. It contains two characteristic secondary structure features (i) a central T-shaped structure (the dimer linkage structure) (ii) two loops symmetrically positioned on each side of the dimer linkage structure. Melting of the dimer linkage structure resulted in a concomitant dissociation of the 52S molecule into two half-size subunits, each about 8-10 Kb. PolyA mapping by electronmicroscopy shows that the "50-60"S molecules contain two polyA segments, one at each end. These results suggest that the "50-60"S RNA consists of two 8-10 Kb or "30-40"S subunits, each with a characteristic secondary structure loop. These two subunits which are possibly identical are joined at their 5' ends within the dimer linkage structure. The primary nucleotide sequence, the molecular weight and the stability of the "50-60"S RNA is different for each different virus. Yet, the finding that these different viral RNA's contain similar secondary structure patterns suggests that such features are functionally important. If this is true, it is possible that they are present in all type C virus genomes. In part II, an electronmicroscopic technique for studying single-stranded RNA is described. This technique has been applied to determine the molecular weight, to study the secondary structure and to map the polyA sequence of RNA isolated from an arbovirus (Sindbis). This spreading technique utilizes glyoxal as a denaturing agent. Glyoxal reacts preferentially with guanine residues of polynucleotides and blocks their hydrogen binding donor functions. Single-stranded RNA, after treatment with glyoxal, appears as an extended filament whose length can be accurately measured by electronmicroscopy. By this means, the molecular weight of Sindbis virus RNA is determined to be 4.7 ± 0.4 x 106 daltons. Glyoxal treatment is useful for the electronmicroscopic mapping of polyA sequences on RNA molecules, since the RNA is extended without affecting the polydT binding which is the basis for polyA mapping. Using this method, a polyA sequence has been mapped at one end of Sindbis virus RNA. Many circular molecules are seen when Sindbis RNA is treated for only short periods with glyoxal, then spread for electron microscopy. Under more denaturing conditions, linear molecules are seen. It is proposed that the two ends of Sindbis viral RNA contain mutually complementary sequences which normally are base-paired to form circular molecules. Under the more denaturing conditions hydrogen bonding is disrupted, which produces linear molecules.