Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T1, and modification with bisulfite.
Potato spindle tuber viroid (PSTV), a small infectios RNA, has been completely digested with RNase T1 and RNase A, and the resulting oligonucleotides have been sequenced using 5'-terminal 32p-labelling with gamma-32p ATP and T4 polynucleotide kinase, fingerprinting and controlled nuclease P1 digestion. Modified nucleotides have not been detected in 5'-positions of these oligonucleotides. PSTV consists of about 359 nucleotides and contains a remarkable stretch of 18 purines, mainly adenosines; there is no AUG initiation triplet present. The established oligonucleotide sequences preclude a perfect intramolecular base complementarity within the covalently closed viroid circle. Therefore, the rigid, rod-like native secondary structure of PSTV, as seen in the electron microscope, must be based on a defective rather than on a homogeneous RNA helix. The detailed analysis of the bisulfite-catalized modification of cytidine to uridine in PSTV revealed a higher reactivity for the majority of the cytidines than would be expected for a perfect helix. Since only cytidines in single-stranded regions are knonw to be fully reactive, this finding provides additional evidence for defects in the helical secondary structure of PSTV.
D001483:Base Sequence; D003562:Cytidine; D008956:Models, Chemical; D009841:Oligonucleotides / Q000032:analysis; D010942:Plant Viruses / Q000032:analysis*; D012328:RNA Viruses / Q000032:analysis; D006163:Ribonuclease T1 / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013447:Sulfites; D014529:Uridine
D009841:Oligonucleotides; D013447:Sulfites; D003562:Cytidine; D012260:Ribonucleases; D006163:Ribonuclease T1; D014529:Uridine