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 electron microscopy. 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 posit1ons 0.57, 0.88, 0.77, and 0.40 to 0.50 of the DNA map. The R loop at position 0.57, 0.88, 0.77, and 0.40 containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these two 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, two locations at which adjacent mRNA's 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.

Early gene expression of adenovirus type 2: R-loop mapping of mRNA and time course of viral DNA, mRNA, and protein synthesis

“…The hybridization of early Ad2 mRNA with Ad2 DNA and the visualization of R loops in the electron microscope are described in the accompanying article (28). In all of the hybridizations of this report, the DNA concentration was 10 ,ug/ml and the RNA concentration was 100 ,tg/ml.…”

“…RNA was extracted from the cytoplasm at different times after infection, and purified on oligo(dT)-cellulose (3) to select for poly(A)-containing Ad2 mRNA (33). R loops were generated under conditions defined previously (28,46) and examined in the electron microscope. Figure 2 summarizes observations of Ad2 DNA molecules carrying early Ad2 mRNA extracted at 2, 4, 6, or 8 h after infection.…”

“…Cells and virus stocks. The propagation and infection of KB cells with purified virus is described in the accompanying article (28).…”

“…Purification and electron microscopy of early Ad2 mRNA. Poly(A)-containing RNA was extracted at various times after infection from the cytoplasm of cells incubated in the presence of cycloheximide (10 ,ug/ml) as previously reported (9,11,28).…”

“…The time of appearance of individual viral mRNA's was compared to the time course of viral protein and DNA synthesis. We present a refined map of adenovirus gene functions which is based on results documented in this and the accompanying study by Meyer et al (1977), as well as on data published by other laboratories. The intricate control system of initial adenovirus type 2 (Ad2) gene expression assigns a number of early viral functions to individual classes of mRNA, transcribed from distinct regions of the genome (5, 7) which comprise noncontiguous parts of either viral DNA strand (32,38).…”