Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA

Abstract

R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by EM. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near positions 0.57, 0.88, 0.77 and 0.40-0.50 for the DNA map. The R loop at position 0.57 corresponded to a region containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these 2 R loops paralleled those of the encoded gene products. The mRNA sizes, calculated from those of the respective R loops, were slightly larger than needed to code for these polypeptides. Using the R-loop technique, 2 locations at which adjacent mRNA hybridized to different strands were accurately mapped at positions 0.61 and 0.91 of the DNA. The map positions of late Ad2 mRNA correlated well to published RNA and protein maps.