Molecular and General Genetics MGG

, Volume 242, Issue 5, pp 495–504 | Cite as

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

  • Rainer Schubbert
  • Clarissa Lettmann
  • Walter Doerfler
Original Articles

Abstract

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.

Key words

Phage M13 DNA Dot blot hybridization Southern blot hybridization Polymerase chain reaction 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Behn-Krappa A, Hölker I, Sandaradura de Silva U, Doerfler W (1991) Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences. Genomics 11:1–7Google Scholar
  2. Boom R, Sol CIA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, van der Noorda J (1989) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28:495–503Google Scholar
  3. Clewell DB, Helinski DR (1972) Effect of growth conditions on the formation of the relaxation complex of supercoiled ColEl deoxyribonucleic acid and protein in Escherichia coli. J Bacteriol 110:1135–1146Google Scholar
  4. Doerfler W (1968) The fate of the DNA of adenovirus type 12 in baby hamster kidney cells. Proc Natl Acad Sci USA 60:636–643Google Scholar
  5. Doerfler W, Gahlmann R, Stahel S, Deuring R, Lichtenberg U, Schulz M, Eick D, Leisten R (1983) On the mechanism of recombination between adenoviral and cellular DNAs: the structure of junction sites. Current Topics Microbiol Immunol 109:193–228Google Scholar
  6. Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  7. Groneberg J, Brown DT, Doerfler W (1975) Uptake and fate of the DNA of adenovirus type 2 in KB cells. Virology 64:115–131Google Scholar
  8. Grunstein M, Hogness DS (1975) Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965Google Scholar
  9. Hofschneider PH (1963) Untersuchungen über kleine E. coli KIZ Bakteriophagen. Z f Naturforsch 18 b: 203–210Google Scholar
  10. Jessberger R, Heuss D, Doerfler W (1989) Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences. EMBO J 8:869–878Google Scholar
  11. Maturin L Sr, Curtiss R III (1977) Degradation of DNA by nucleases in intestinal tract of rats. Science 196:216–218Google Scholar
  12. McAllan AB (1980) The degradation of nucleic acids in, and the removal of breakdown products from the small intestines of steers. British J Nutr 44:99–113Google Scholar
  13. McAllan AB (1982) The fate of nucleic acids in ruminants. Proc Nutr Soc 41:309–317Google Scholar
  14. Orend G, Kuhlmann I, Doerfler W (1991) Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells. J Virol 65:4301–4308Google Scholar
  15. Orend G, Linkwitz A, Doerfler W (1994) Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns. J Virol 68:187–194Google Scholar
  16. Pääbo S, Gifford JA, Wilson AC (1988) Mitochondrial DNA sequences from a 7000 year old brain. Nucleic Acids Res 16:9775–9787Google Scholar
  17. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich H (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491Google Scholar
  18. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  19. Storm E, Brown DS, Orskov ER (1983) The nutritive value of rumen micro-organisms in ruminants. 3. The digestion of microbial amino and nucleic acids in, and losses of endogenous nitrogen from, the small intestine of sheep. British J Nutr 50:479–485Google Scholar
  20. Sutter D, Westphal M, Doerfler W (1978) Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells. Cell 14:569–585Google Scholar
  21. Tatzelt J, Fechteler K, Langenbach P, Doerfler W (1993) Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site. Proc Natl Acad Sci USA 90:7356–7360Google Scholar
  22. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Rainer Schubbert
    • 1
  • Clarissa Lettmann
    • 1
  • Walter Doerfler
    • 1
  1. 1.Institute of GeneticsUniversity of CologneCologneGermany

Personalised recommendations