FATE OF FOOD-INGESTED FOREIGN DNA IN THE GASTROINTESTINAL TRACT OF MICE
The tempting interpretation that DNA methylation, particularly the de novo methylation of integrated foreign DNA, is part of an ancient cellular defense mechanism raises a number of questions. One of the obvious ones relates to the major origins of foreign DNA, e.g. in mammals. Virus infection as such a contingency has been extensively discussed in this chapter. Another apparent source of large amounts of foreign DNA all organisms are constantly exposed to is the DNA orally ingested with the food supply. We have therefore undertaken a study on the fate of food-ingested foreign DNA in mice as model organism. I will present a short summary of the major results my laboratory adduced in a project which we initiated in 1988.
In mammals, the gastrointestinal tract is the main portal of entry for foreign macromolecules, and its epithelial lining presents the immediate sites of contact with foreign DNA and proteins. In our investigations on the fate of foreign DNA in the digestive tract, we fed naked test DNAs of various derivations to laboratory mice at between two and six months of age [132-134]. The DNA of bacteriophage M13, the DNA of human adenovirus type 2, or the gene for the green fluorescent protein (GFP) from Aequorea victoria were administered as test DNAs in different experiments. None of these DNAs had homologies to bacterial or mouse DNA, except for perhaps very short stretches of DNA sequence, which were then excluded from being used for the detection of the foreign DNA in the mouse organism.
In later experiments, we fed leaves of the soybean plant to mice and followed the fate of the strictly plant-specific Rubisco (ribulose 1,5-diphosphate carboxylase) gene. During the passage through the gastrointestinal tract of mice, the bulk of the administered DNA is completely degraded. However, a few percent of the test DNAs resist the digestive regimens of the gut and can be recovered for several hours after feeding in various parts of the intestinal tube as fragments between 1700 nucleotides (nt) (rare) and a few 100 nt. By applying a variety of techniques (Southern blotting, polymerase chain reaction (PCR), FISH, and rescue of the test DNA fragments by recloning and resequencing) the test DNAs could be followed to the wall of the intestinal tract, particularly the colon, to Peyer patches, peripheral white blood cells, and to cells of the liver and spleen [132-136]. When pregnant animals were test DNA fed, fragments of the test DNA could be traced by FISH and PCR to clusters of cells in various organs of the embryo, but never to all its cells. Moreover, when mice were fed daily and continuously for eight generations, transgenic animals were never observed. Hence, we assume that the germ line must be protected from the exposure to and the uptake of food-ingested foreign DNA. Moreover, we never obtained evidence for the test DNA being transcribed in any of the organ systems of the adult animals, which had been given test DNA [135]. The possible transcription of test DNA was assessed by RT-PCR, the most sensitive technique to detect trace amounts of specific transcripts. After feeding mice daily for one week, test DNA could be recloned, however extremely rarely, from the spleen of the animals. In a few of these clones, mouse specific DNA was found adjacent to the test DNA in the cloned DNA. Further proof will be required to investigate the possibility of whether foreign DNA could be integrated into the genome of defense cells in the recipient animals [133]. In a completely independent approach, we could demonstrate that the protein glutathione-S-transferase, a rather stable protein, survived in the stomach and small intestine of mice for up to 30 min after feeding [136].
Taken together, the results of this series of investigations indicate that foreign macromolecules, particularly the very stable DNA, can survive in the gastrointestinal tract at least transiently in small amounts and in fragmented form and can get access to various organ systems of the mouse. Even stable proteins survive only for a very short time in the gastrointestinal tube. We have not found any evidence for the entry of foreign DNA into the germ line, nor could we demonstrate transcription of foreign DNA in any of the organ systems tested. It is not known whether a tiny proportion of the thus persisting DNA may find entry into the genome of a rare defense cell and remain there with unknown functional consequences. These questions will be worth pursuing.
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