An analysis by electron microscopy of early simian virus 40 RNA from a tsA mutant

Abstract

A new electron microscopic technique, developed by J. Ferguson and R. W. Davis (manuscript in preparation), has been used to investigate the pattern of early transcription in cells infected with simian virus 40 (SV40). DNA binding protein from E. coli and antibody to this protein increase the apparent thickness of single-stranded DNA. Thus the positions of termini and the sizes of individual molecules of RNA can be determined after hybridization to a defined single strand of DNA. Since the concentration of early RNA is very low in cells infected by wild-type SV40, the RNA was prepared from cells infected with the temperature-sensitive mutant tsA58, where early RNA is greatly over-produced after a shift from permissive to restrictive temperature late in infection. Nuclear or cytoplasmic RNA was hybridized to full-length linear early strands of SV40 DNA. Using the known end points generated by the restriction endonuclease (Eco RI) and an internal marker to define orientation of the strand, the positions of the 5′ and 3′ termini of individual RNA molecules were determined. Most molecules of cytoplasmic early RNA have a 5′ terminus at 0.67 SV40 fractional length, and the majority of the 3′ termini extend well into the late region, at least as far as the Eco RI restriction site at map position 0. The mean 5′ terminus of nuclear early RNA molecules was at 0.68 SV40 fractional length. Many of these molecules are small (0.01–0.20 fractional length), but some extend to the Eco RI restriction site or possibly beyond, as found for the cytoplasmic early RNA. The effects on the composition and size of early RNA pools of the tsA mutation or of exposure to high temperature are unknown.