Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules

Abstract

We have synthesized a convenient electron microscope label for mapping poly(A) sequences. Short lengths of poly(dT) are polymerized onto nicked circular SV40 DNA with the enzyme terminal deoxynucleotidyl transferase. An RNA or DNA molecule of interest is treated with glyoxal, hybridized briefly with the poly(dT) circles, and spread for microscopy; poly(A) stretches are clearly marked because they are attached to the poly(dT) on the easily recognized SV40 duplex circles. The RNAs of several type C oncornaviruses were examined by this method. The endogenous feline virus(RD-114), the endogenous baboon virus (BKD), and the woolly monkey sarcoma virus (WoMV) all contain a dimer of RNA subunits held together in a central secondary structure feature we call the dimer linkage structure. Both ends distal to the dimer linkage structure hybridize to the SV40-poly(dT). Assuming both poly(A)s are on the 3' ends of the subunits and that both subunits are identical, the two identical subunits are held together by interactions between sequences close to the 5' ends.