Quantitative electron microscopy of early adenovirus RNA.

Abstract

Abstract We present methods of electron microscopy which allow simultaneous mapping and quantitation of individual populations of RNAs which are complementary to distinct regions of one template DNA. Adenovirus transcription, which has been thoroughly studied in the past, was chosen as a test system for our procedures. The cytoplasm of human cells harvested at early stages of productive infection with adenovirus type 2 contains RNA which hybridizes to four well defined regions of the viral genome, two on each DNA strand. We annealed the RNA to the separated strands of virion DNA. Hybrids (RD molecules) were visualized either directly or after duplexing with the complementary DNA strand (HD molecules). The four early regions were mapped between positions 1.1 to 10.6, 61.6 to 68.1, 76.7 to 83.7, and 91.5 to 96.9. At six and at eight hours after infection, RNAs were observed in these four locations at relative abundances of 1:1.62: 2.60:1.35, and 1:1.54:2,46:1.27, respectively. At least five distinct populations of RNA have been discerned, two in region 1, and one in each of the other regions. Special features detected in the electron microscope include a loop of single stranded DNA bridged by RNA at position 77.8 79.0 and a tiny denaturation bubble within HD molecules near position 29. Both structures may have interesting biological implications. All map positions represent mean values, with standard deviations of ~60 nucleotides in the best and of ~600 nucleotides in the worst cases, on a map comprising ~35,000 nucleotides. Individual transcripts contained in nanogram amounts of crude preparations may be assayed on single or double-stranded templates by our procedures. The experiments are more precise and less time consuming than comparable biochemical studies. They should be amenable to a variety of biological systems dealing with templates that are of, or can be restricted to, a size suitable for electron microscopy.