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Food-ingested foreign DNA is not completely degraded in the gastrointestinal tract of mice. Phage M13mp18 DNA as a test molecule devoid of homology to mouse DNA was pipette-fed to or added to the food supply of mice. The fate of this foreign DNA in the animals was followed by several methods. In 84 animals, fragments of M13mp18 DNA were detected in the contents of the small intestine, the cecum (until 18 h), the large intestine, or the feces. In 254 animals, M13mp18 DNA fragments of up to 976 bp were found in blood 2-8 h after feeding. In buffer-fed control animals, M13mp18 DNA could not be detected. M13mp18 DNA fragments were traced by PCR in peripheral leukocytes and located by f luorescent in situ hybridization in about 1 of 1000 white cells between 2 and 8 h, and in spleen or liver cells up to 24 h after feeding, but not later. M13mp18 DNA could be traced by f luorescent in situ hybridization in the columnar epithelial cells, in the leukocytes in Peyer's patches of the cecum wall, in liver cells, and in B cells, T cells, and macrophages from spleen. These findings suggest transport of foreign DNA through the intestinal wall and Peyer's patches to peripheral blood leukocytes and into several organs. Upon extended feeding, M13mp18 DNA could be recloned from total spleen DNA into a vector. Among about 2.5 ؋ 10 7 plaques, one plaque was isolated that contained a 1299 nucleotide pair fragment (nt 4736-6034) of sequence-identified M13mp18 DNA. This fragment was covalently linked to an 80 nt DNA segment with 70% homology to the mouse IgE receptor gene. The DNA from another plaque also contained mouse DNA, bacterial DNA, and rearranged DNA. Two additional plaques contained M13mp18 DNA fragments of at least 641 (nt 2660-3300) or 794 (nt 4640-5433) nucleotide pairs. The medical and evolutionary implications of these observations may be considerable.

Dinucleotide overand underrepresentation is evaluated in all available completely sequenced DNA or RNA viral genomes, ranging in size from 3 to 250 kb (available RNA viruses fall into the small-virus category). The dinucleotide CpG is statistically underrepresented (suppressed) in all but four of the small viruses (more than 75 with lengths of <30 kb) but has normal relative abundances in most large viruses (-30 kb). Most retrotransposons in eukaryotic species also show low CpG relative abundances. Interpretations, especially in some cases of DNA viruses or viruses with a DNA intermediate, might relate to methylation effects and modes of viral integration and excision. Other possible contributing factors relate to dinucleotide stacking energies, special mutation mechanisms, and evolutionary events.

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We have investigated DNA methylation in human Alu sequences, both in general and in specific Alu sequences associated with the genes for a1 globin, tissue plasminogen activator (tPA), adrenocorticotropic hormone (ACTH) and angiogenin. We studied DNAs from lymphocytes, granulocytes, brain, heart muscle and sperm, and from the human HeLa and KB cell lines by using cleavage with methylation-sensitive restriction enzymes combined with Southern blot hybridization and by using genomic sequencing. The results can be summarized as follows. (i) In differentiated primary human cells, Alu elements are often highly methylated even when they are in very 5'-CG-3'-rich regions. This finding is not consistent with the notion that hypermethylation would be a sufficient condition in itself for 5'-CG-3' sequences to undergo loss of 5-methyldeoxycytidine (5-mC) due to deamination and subsequent mutation. (ii) There are distinct differences in the levels of methylation in the specific Alu sequences. (iii) Alu elements in the DNA of haploid spermatozoa are much less methylated than in diploid cells. Preliminary data indicate that spermatozoa contain Alu-specific RNAs. (iv)The results of cell-free transcription experiments with Alu elements suggest that the in vitro transcription of Alu elements can be inhibited by 5'-CG-3' methylation. High levels of 5'-CG-3' methylation in Alu elements could contribute to their general transcriptional inactivity. (v) The patterns of methylation observed in the Alu elements and in the surrounding sequences are characterized by cell type specific interindividual concordance.

The adenovirus type 12 (Adl2) DNA sequences integrated into the DNA of four lines of Adl2-transformed hamster cells are extensively methylated. Methylation in mammalian cell DNA is believed to occur predominantly at 5'-C-G-3' sequences. The majority, although not all, of the 5'-C-C-G-G-3 sequences present in integrated Adl2 DNA are methylated. Ad12 DNA isolated from purified virions, on the other hand, is not methylated to any significant extent.

Adenovirus type 12 (Adl2) is oncogenic in neonate hamsters (1, 2), and the viral genome can chromosomally integrate into the genome of hamster cells (3-7). Adl2 DNA insertion is not site-specific; transcriptionally active cellular DNA sequences seem to be preferred targets (8, 9). The molecular mechanism of Adl2 DNA insertional recombination is akin to nonhomologous recombination, as studied in a cell-free system (10, 11). We have pursued the possibility that foreign (Adl2) DNA insertion contributes to the process of tumorigenic transformation by Adl2. The conventional notion of insertional mutagenesis disrupting cellular DNA at the immediate locus of foreign DNA integration has been extended to the concept that cellular gene activity could be affected at many different cellular sites close to or remote from the locus offoreign DNA insertion due to changes in DNA methylation patterns (5, 6, 12).In Adl2-transformed hamster cells, in Adl2-induced hamster tumor cells, and in hamster cells carrying integrated Adl2The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5515 genomes, integrated plasmid, or bacteriophage A DNA without transformed phenotype, the methylation of several cellular DNA segments is markedly enhanced in comparison with BHK21 or primary hamster cells. The randomly selected hamster cellular DNA probes used in methylation analyses have been localized by fluorescent in situ hybridization (FISH) to different hamster chromosomes that do not carry Adl2 DNA. Thus, integration of foreign DNA in hamster cells exerts a distinct trans effect on the methylation of several cellular DNA segments located on different chromosomes.