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. 2021 Jun 1;302:198466. doi: 10.1016/j.virusres.2021.198466

Fig. 1.

Fig. 1

Foreign DNA insertion into hamster genome alters profiles of cellular DNA methylation in the ~ 900 copies of IAP retrotransposon sequences. Altered methylation patterns are maintained even after loss of the transgenomes.

[A] In situ hybridization of Ad12-transformed hamster cell nucleus of a T637 cell visualizes about 10 to 12 copies of chromosomally integrated Ad12 DNA (Ad12 DNA probe, green, white arrow); IAP probe (pink) ~ 900 copies of Intracisternal A Particle retrotransposon genomes that are frequently located on short arms of hamster chromosomes; DAPI-staining (blue) of cellular DNA.

[B] Southern blot hybridization of DNAs from (right to left) Ad12 (human adenovirus type 12); T637 (Ad12-transformed BHK21 hamster cells); BHK21 (baby hamster kidney cell line); T637; TR3 (a revertant of T637 cell line whose genome had lost all of the originally 10 to 12 integrated viral genome copies (PCR-confirmed); A2497-3 is an Ad12-transformed hamster cell line different from T637 cells; BHK˖Ad12, BHK21 hamster cells abortively infected with Ad12 (Doerfler, 1969, Hochstein et al., 2008). The 32P-labeled DNA hybridization probes were designated at the bottom of the electropherogram, Ad12 or IAP (from right to left).

Levels of CpG DNA methylation in the different DNA samples were assessed by differential cleavage with methylation-sensitive (HpaII, HhaI) or methylation-insensitive restriction endonucleases (MspI): MspI does cleave 5´-CCGG-3´ and 5´-CmCGG-3 sequences (methylation-insensitive), HpaII and HhaI (methylation-sensitive) do not cut 5´-CmCGG-3´ sites. The results document distinct increases of CpG methylated sequences in the Ad12-transgenic cell lines T637 and A-2497-3 as evidenced by the large amounts of HpaII- and HhaI-uncut IAP DNA (top of display). The revertant cell line TR3, derived from T637 cells, also shows increased IAP DNA methylation, although it had lost all copies of the Ad12 transgenomes (PCR confirmed). Apparently, the epigenetic effect (increased IAP DNA methylation) of transgene insertion on the IAP retrotransposon DNA persisted even after the loss of the transgenes that had elicited the trans epigenetic effects. These figures were taken from (Heller et al., 1995).