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3-Food: More on "gut reaction"



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by Henning Strodthoff, GeN
see mail: 3-Food: genes for antibiotic resistance could jump [08.02.1999]

>conclusion
There is no scientific evidence for the uptake of free DNA in the gut 
till now.
The Research-team at TNO showed, 
   - that free DNA is pretty stable in the large intestine.
   - DNA from gm Tomato can reach the "safe" large intestine.
   - These results suggest, that free DNA is available for uptake in 
     the large intestine.
   - Transformation of bacteria by DNA from gm tomato in the large 
intestine 
     was shown to be very unlikely 

>Zusammenfassung: 
Bisher liegen keine wissenschaftlichen Beweise fuer die Aufnahme freier 
DNA (durch Bakterien) im Darm vor. Das Forschungsteam am TNO konnte 
zeigen,
    - das freie DNA im Dickdarm relativ stabil ist.
    - DNA aus gtv Tomaten kann diesen "sichern" Bereich des Darms 
erreichen.
    - Diese Ergebnisse legen nahe, das freie DNA fuer die Aufnahme durch 
Bakterien
      zur Verfuegung steht.
    - Die Aufnahme von DNA aus gtv Tomaten durch Bakterien erscheint 
dennoch unwahrscheinlich, 
      so Jos M.B.M. van der Vossen, ein Forscher am TNO.
Die folgenden Notizen sind nach einem Gespraech mit Jos M.B.M. van der 
Vossen, einem Forscher am TNO entstanden und von ihm gegengelesen.  



_________________
Dear friends
In the following some notes from a phonecall with Jos M.B.M. van der 
Vossen from TNO, who did the research with the "artificial gut" (=> 
NewScientist: gut reaction)

Actually he is pretty unhappy with the articel published in the 
NewScientist. Here you see what they did:

>free DNA
Free DNA was digested very quickly in the System (Stomach, Duodeum, 
Jejunum, Ileum). After 10 sec it was no longer detectable.
BUT
Free DNA is much more stable in the "large intestine". Even after 5 to 6 
min the DNA was detectable.
It was not possible to show any uptake of this free DNA by any bacteria.

>gm tomato
Wenn they put crushed gm Tomato into the System: 5 to 10 % of the DNA 
(packed in tomato-cells) passed the first part of the System (Stomach, 
Duodeum, Jejunum, Ileum) and entered the "large intestine".
It was not possible to show any uptake of this DNA by any bacteria.

>Problems
Untill now, it was not possible to show any uptake of DNA. So they 
dicided to make a "worst case" experiment: They introduced a broad host 
range plasmid into the microflora of the in vitro large intestine. In the 
experiment they hoped to find bacteria that were transformed by the 
plasmid. 

They used bacteria with gm Plasmid-DNA. In the experiment they hoped to 
find bacteria-to-bacteria DNA-exchange.
Even in this experiment it was not possible to show any uptake of DNA by 
bacteria.
Therefore they analysed to what extend bacteria could be detected in this 
complex ecosystem. E. coli containing a marker gene was released at 
different levels in the complex flora. From this experiment it appeared 
that only 1 E. coli cell could be detected among approximately 10E9 
bacteria in the large intestine by using nested PCR detection. 

Thus, the introduction of the broad host range plasmid resulted in a 
transformation event with a rate lower than 1 / 10E9. These data  
indicate that transformation of bacteria by marker genes from the 
genetically modified tomato in the microflora is very unlikely. Because: 
1) the marker gene needs to be integrated in the bacterial genome 
2) there are no homologeous sequences available for integration
3) the marker gene lacks appropriate expression signals 
So, Transformation is very unlikely. 

>Gene transfer from bacterium to bacterium by conjugation:
DNA could only be transfered from bacterium to bacterium via conjugation. 
This was shown in vitro as was shown previously in mice. For this, "we" 
introduced the donor cells of Enterococcus faecalis containing the broad 
host range plasmid pAMss1 into the in vitro large intestine together with 
recipient cells. Even in the presence of a complex microflora transfer of 
pAMss1 was observed with low frequency (1/10E7). 
Donor cells of bacteria that are unable to grow or to compete in the 
faecal flora were unable to transfer their conjugative broad host range 
plasmid to the potential recipient strains. 




Best wishes

Henning Strodthoff, GeN

_________________
enclosed the adress of
>TNO
Jos M.B.M. van der Vossen, PhD

Product Manager Biomonitoring
Microbiology and Quality Management Division

TNO Nutrition and Food Research Institute
P.O. Box 360            Utrechtseweg 48
3700 AJ Zeist            3704 HE Zeist
The Netherlands

Phone: + 31 30 69 44 720
Fax:      + 31 30 69 57 901
E-mail: vandervossen@voeding.tno.nl
Internet: http://www.voeding.tno.nl
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