Transgenic mice were generated with pRSV-CAT, a chimeric gene construct containing the long terminal repeat of Rous sarcoma virus (RSV) linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT expression, detected in adult animals of five independent strains, was preferentially directed to organs rich in tendon, bone, and muscle. This pattern reflects the disease specificity of the intact virus and suggests that the tissue tropism of RSV is determined at least in part by the presence of endogenous tissue-specific factors that can promote expression of genetic information linked to the long terminal repeat. In two of the mouse strains, insertion of the pRSV-CAT DNA resulted in developmental abnormalities. One of these strains was characterized by a dominant trait of embryonic lethality, the other by a recessive trait of fused toes in all four feet.

Separated strands of adenovirus DNA were annealed with ear1y cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, sin-tranded DNA. Loops have been observed and mapped in al four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression. The synthesis of mRNA in eukaryotic cells consists of a number of distinct but poorly understood steps. After transcription from the template DNA, the RNA transcript appears to undergo a series of nucleolytic cleavages, modification of the 5' end to form the "cap" structure, and addition of poly(A) to the 3' end (1, 2). We have been studying the RNAs synthesized after infection of KB cells by human adenovious type 2 (Ad2) in an attempt to understand these processes. The Ad2 system provides an excellent model in which to study these processes in that its DNA is far less complex than that of a eukaryotic cell and has few gene products but appears to share the same mechanism for RNA transcription and processing.The viral gene products can be placed into two separate classes: those produced early during infection (that is, before the onset of viral DNA synthesis); and those produced after its onset (late RNAs). We have mapped early and late cytoplasmic RNAs (3-5) as well as late nuclear RNAs (6) by using the R-loop electron microscope technique (7,8). In addition, use of quantitative electron microscopy has enabled us to determine the relative abundance of individual populations of cytoplasmic Ad2 RNA (9, 10). Hybridization of RNA with separated strands of Ad2 DNA revealed structures reminiscent of insertions previously observed in Drosophila melanogaster rDNA'RNA hybrids (7,11,12 RESULTSEarly cytoplasmic Ad2 RNA hybridizes to four regions of the genome (14, 15) which we (10) located at map positions 1.1-10.6, 61.6-68.1, 76.7-83.7, and 91.5-96.9. The rightward DNA strand contains regions 1 and 3; the leftward strand contains regions 2 and 4. Fig. 1A depicts a rightward Ad2 DNA molecule that carries RNA in region 1 (left end) and in region 3 (right of center). Arrows point to small loops of single-stranded nucleic acid that protrude, at two locations, from the DNA-RNA duplex structures. We have also observed loops in regions 2 and 4 on the leftward strand and a second loop occurring at a different site in region 1. Fig. 1 B and C shows enlargements of the two loops found in region 1. These RNAs have been mapped at positions 1.2-5.5 (region la) and 4.4-11.3 (region lb) with the loops mapping between points 2.9 and 4.0 and 5.9 and 9.7, respectively. The 3' terminus of region la RNA (5.5) overlaps with the 5' terminus of region lb (4.4) RNA. Both of these loops occur infrequently, with RNAs not generating a loop and mapping between positions 1.1 and 10.6 being the predominant RNAs found in this region.Two single-stranded DNA displacement loop...

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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.