Expression of Viral DNA in Adenovirus Type 12-Transformed Cells, in Tumor Cells, and in Revertants

Sabine Schirm, Walter Doerfler
1981 Journal of Virology  
The expression as cytoplasmic RNA of integrated human adenovirus type 12 (Adl2) DNA in transformed and tumor cell lines and in revertants was investigated. The transformed and tumor cells contained multiple copies of the viral genome, 3 to 22 copies per cell in different cell lines. The integrated Adl2 DNA molecules persisted intact or nearly intact and in most cases colinear with the virion DNA. In the revertant cell lines, which were derived from cell line T637 (22 copies of Adl2 DNA per
more » ... , all of the Adl2 DNA molecules were lost (line F10) or only one copy and a fraction of a second copy persisted (line TR12). The size classes and map locations of Adl2-specific cytoplasmic RNAs in three Adl2transformed hamster cell lines (T637, HA12/7, and A2497-3), in two revertant lines (FlO and TR12), in one Adl2-induced hamster (CLAC3), and in one rat brain tumor line (RBT12/3) were determined. Cytoplasmic RNA from uninfected B3 hamster cells and from human KB cells productively infected with Adl2 served as controls. In the latter control experiments, the RNA was isolated early or late postinfection. With respect to the amounts of Adl2-specific RNAs detected in cytoplasmic RNA from various Adl2-transformed or Adl2-induced tumor cell lines, we could not establish any correlations to the number of Adl2 genome copies integrated into the cellular DNAs. Thus, the expression of the integrated viral genomes in these lines was regulated by mechanisms more complicated than simple gene dosage effects. Using cloned fragments of Adl2 DNA as hybridization probes, we analyzed the cytoplasmic RNAs from the cell lines mentioned by electrophoresis on agarose gels, blotting, and DNA-RNA hybridization. For each transformed and tumor cell line, except for the revertants, several size classes of Adl2-specific cytoplasmic RNA were detected for the early El, E2, and E4 regions of Adl2 DNA. Some of these size classes were similar but not identical to those observed in cytoplasmic RNA isolated early from human KB cells productively infected with Adl2. Only cell lines A2497-3, T637, and RBT12/3 contained several size classes of cytoplasmic RNA homologous to the E3 region of Adl2 DNA. Weak homologies to the El region of Adl2 DNA were also detected in the revertant lines F10 and TR12. Late regions of Adl2 DNA were expressed as cytoplasmic RNA in cell lines CLAC3 and RBT12/3. Weak homologies were detected between certain segments of the Adl2 genome (the EcoRI-B, -C, and -D fragments) and the cytoplasmic RNA from uninfected hamster cells. These homologies had no apparent counterpart at the level of DNA, perhaps because these homologies could be detected only due to an overrepresentation of RNA sequences. In preliminary experiments, we failed to detect the expression as cytoplasmic RNA of the so-called virus-associated RNA in transformed and tumor cell lines. Virus-associated RNA represents a population of low-molecularweight RNAs that map at around 30 fractional length units on the viral genome. The pattems of integration and persistence of tures emerge with respect to the integration viral DNA in adenovirus type 12 (Adl2)-trans-patterns of Adl2 DNA. Multiple copies of the formed hamster cells and of Adl2-induced ham-Adl2 genome are integrated in complex arrangester and rat tumor cells have been investigated ments. A portion of the Adl2 DNA molecules is in detail (8, 9, 13, 22, 26 ; I. Kuhlmann and W. inserted colinearly with virion DNA. However, Doerfler, submitted for publication). Although within sequences close to the termini of the each transformed and tumor line appears to Adl2 DNA, selective amplifications have been have its specific properties, certain general fea-observed (22). Moreover, the persistence ofAdl2 694 on May 8, 2020 by guest
doi:10.1128/jvi.39.3.694-702.1981 fatcat:v7fnoat7yfd4rgif5pqros6oy4