BACKGROUND
artificial dna has been used for hiding messages or for data storage  <cit> .
dna-crypt uses redundant ranges in the genetic code to introduce a watermark in a coding region.
amino acid codes are redundant so the watermark can be integrated by changing these dna triplets.
dna-crypt checks for synonymous codons in the genome and replaces the bases at the third position with a new base, which encodes parts of the watermark.
the algorithm can be combined with other encryption algorithms like rsa, aes or blowfish  <cit> .
mutations, which can occur will be corrected by dna-crypt itself using several mutation correction codes like the hamming-code or the wdh-code  <cit> .
an integrated fuzzy controller decides on a set of heuristics, whether to use a correction code or not for optimal performance.
in silico studies using the ypt <dig> gene of saccharomyces cerevisiae showed that inserting these watermarks into a coding region did not affect the translation of proteins  <cit> .
searching for a homologous protein to mammalian rab <dig> in saccharomyces cerevisiae, ypt <dig> was first discovered in  <dig> by wichmann et al.
<cit> .
the ypt <dig> gene encodes a  <dig> amino acid protein of  <dig>  kda  <cit> .
it is involved in the homotypic vacuolar fusion and essential for the formation of the snare complexes at the vacuolar membrane  <cit> .
in addition ypt <dig> interacts with the hops-complex  and the vam <dig> protein .
a loss of ypt <dig> leads to undocking of the hops-complex and vam7p  <cit> .
the vam <dig> gene was discovered in a screen for saccharomyces cerevisiae mutants, which have defects in the vacuolar morphology  <cit> .
the vam <dig> gene encodes a  <dig> amino acid protein of  <dig>  kda.
strains lacking vam <dig> or ypt <dig> have various vesicular structures instead of distinct vacuoles  <cit> .
vam7p consists of two domains, the px and the snare domain .
so far the px domain has not been found in other snare proteins.
it is thought to be necessary for the transport of vam7p to the vacuolar membrane, whereas the snare domain is essential for the homotypic fusion  <cit> .
the function of vam7p in the sporulation processes of saccharomyces cerevisiae has not been elucidated in detail yet, but it has been shown that Δvam <dig> and Δypt <dig> strains are not able to produce spores  <cit> .
in addition Δvam <dig> strains exhibit a reduced proliferation rate in rich medium   <cit> .
for in vivo studies we used a trp- mutant saccharomyces cerevisiae strain  carrying a defective vam <dig> gene  leading to incomplete vacuolar morphology   <cit> .
in addition the cg <dig> strain is unable to sporulate  <cit> .
as a control we used the parental cg <dig> strain, which carries an intact vam <dig> gene .
some times ago it was shown that transformation of cg <dig> with an intact vam <dig> gene leads to normal vacuolar morphology and rate of sporulation .
RESULTS
to investigate, whether the insertion of a watermark into the coding region of the vam <dig> gene has an effect on the vam <dig> protein, we produced a mutagenized vam <dig> sequence, which we transferred into a yeast strain with an amber mutation within the vam <dig> gene leading to an inoperable gene product.
the analysis of the watermarked dna sequence with the dna-crypt fuzzy controller recommended not to use any correction code.
the calculated mutation rate φ was  <dig> .
a pairwise sequence alignment using clustalw showed that the identity of vam <dig> and the watermarked vam <dig> sequence is  <dig> %   <cit> .
the mutagenized base pairs are localized in the snare domain, which is thought to be essential for the homotypic fusion  <cit> .
the integrated watermark did not influence the function of vam <dig> and the resulting phenotype of the cg <dig> cells transformed with prs <dig> vam7-tb show no significant differences compared to the cg <dig> cells transformed with prs <dig> vam <dig>
the vacuolar morphology in both transformed strains was similar compared to the wild type strain .
the number of vacuoles was slightly increased compared to the wild type strain .
as shown in figure  <dig> cg <dig> cells transformed with prs <dig> vam <dig> and cg <dig> cells transformed with prs <dig> vam7-tb display no significant differences, which points to the fact that the insertion of the watermark has no influence to the resulting protein.
in contrast to strain cg <dig>  which is not able to sporulate most likely due to the lack of a functionally active vam <dig> in cg <dig> cells,  <dig> % ±  <dig>  of the prs <dig> vam <dig> and  <dig> % ±  <dig>  of the prs <dig> vam7-tb transformed yeast cells formed spores.
the spores of the transformed cg <dig> cells displayed the normal phenotype of cg <dig> spores .
also in these experiments the insertion of the watermark did not result in an altered phenotype, comparing cg <dig> cells transformed with prs <dig> vam <dig> and cg <dig> cells transformed with prs <dig> vam7-tb.
cg <dig> cells and the cg <dig> cells transformed with prs <dig> vam <dig> or prs <dig> vam7-tb, respectively, displayed a higher division rate compared to cg <dig> cells .
the division rate  is calculated by
odt9odt0=a <dig>
with measuring points ti, i =  <dig> ..,  <dig>
in addition there are no significant differences comparing the division rates of the cg <dig> cells transformed with prs <dig> vam <dig> and the the cg <dig> cells transformed with prs <dig> vam7-tb .
our aim was to prove that the insertion of the watermark 'tb' into the prs <dig> dna does not influence the expression of a functional vam <dig> gene.
by testing the morphology and several growth parameters we demonstrate that the cells containing prs <dig> or prs <dig> plus the watermark tb are indistinguishable from each other with respect to the parameters verified.
the vam <dig> negative and untransformed wild type cells were included in the experiments as controls.
previously reported in silico studies using the ypt <dig> genes of saccharomyces cerevisiae demonstrated that using watermarks in coding regions did not influence the resulting protein  <cit> .
in this paper we show for the first time that the in silico results could be confirmed in vivo by analyzing living yeast cells.
CONCLUSIONS
to our knowledge the insertion of watermarks into eukaryotic cells has not been reported so far.
only storage of information in bacteria has been published  <cit> .
as prokaryotic and eukaryotic cells show very different complexities e. g. on the levels of transcription, translation or compartmentalization it is very important to test and prove the application of dna watermarks into coding regions of living eukaryotic cells.
and this to our knowledge was successfully done for the first time in the experiments which we report on in this manuscript.
our dna-crypt algorithm clearly represents advances compared to the algorithms reported by wong et al.
or arita and ohashi.
it permits the use of several mutation detection and correction codes, like the hamming-code or the wdh-code and binary encryption algorithms like aes, blowfish or rsa  <cit> .
further it provides the use of one-time pads  <cit> .
additionally the dna-crypt algorithm allows an increased amount of information per base and further has an integrated fuzzy controller that recommends whether to use a specific correction code or not  <cit> .
although, for reasons of economy, we inserted only 'tb' into the vam <dig> gene more than two letters could be integrated without any effect to the resulting protein, because dna-crypt only produces silent mutations.
to introduce longer watermark sequences more expensive synthesis of oligonucleotides and extended mutagenesis procedures would have to be performed.
nethertheless the resulting observations would be the same based on the fact that dna-crypt only produces silent mutations.
our experiments using the vam <dig> gene are a proof of concept for introducing dna watermarks into coding regions and can be generalized for other proteins.
the use of dna watermarks in non coding or regulatory sequences will be subject to further examinations.

 artificial dna has been used for hiding messages or for data storage. dna-crypt uses redundant ranges in the genetic code to introduce a watermark in a coding region. amino acid codes are redundant so the watermark can be integrated by changing these dna triplets. dna-crypt checks for synonymous codons in the genome and replaces the bases at the third position with a new base, which encodes parts of the watermark. the algorithm can be combined with other encryption algorithms like rsa, aes or blowfish. mutations, which can occur will be corrected by dna-crypt itself using several mutation correction codes like the hamming-code or the wdh-code. an integrated fuzzy controller decides on a set of heuristics, whether to use a correction code or not for optimal performance. in silico studies using the ypt gene of saccharomyces cerevisiae showed that inserting these watermarks into a coding region did not affect the translation of proteins. searching for a homologous protein to mammalian rab in saccharomyces cerevisiae, ypt was first discovered in by wichmann et al.. the ypt gene encodes a amino acid protein of kda. it is involved in the homotypic vacuolar fusion and essential for the formation of the snare complexes at the vacuolar membrane. in addition ypt interacts with the hops-complex and the vam protein. a loss of ypt leads to undocking of the hops-complex and vam7p. the vam gene was discovered in a screen for saccharomyces cerevisiae mutants, which have defects in the vacuolar morphology. the vam gene encodes a amino acid protein of kda. strains lacking vam or ypt have various vesicular structures instead of distinct vacuoles. vam7p consists of two domains, the px and the snare domain. so far the px domain has not been found in other snare proteins. it is thought to be necessary for the transport of vam7p to the vacuolar membrane, whereas the snare domain is essential for the homotypic fusion. the function of vam7p in the sporulation processes of saccharomyces cerevisiae has not been elucidated in detail yet, but it has been shown that Δvam and Δypt strains are not able to produce spores. in addition Δvam strains exhibit a reduced proliferation rate in rich medium. for in vivo studies we used a trp- mutant saccharomyces cerevisiae strain carrying a defective vam gene leading to incomplete vacuolar morphology. in addition the cg strain is unable to sporulate. as a control we used the parental cg strain, which carries an intact vam gene. some times ago it was shown that transformation of cg with an intact vam gene leads to normal vacuolar morphology and rate of sporulation.  to investigate, whether the insertion of a watermark into the coding region of the vam gene has an effect on the vam protein, we produced a mutagenized vam sequence, which we transferred into a yeast strain with an amber mutation within the vam gene leading to an inoperable gene product. the analysis of the watermarked dna sequence with the dna-crypt fuzzy controller recommended not to use any correction code. the calculated mutation rate φ was. a pairwise sequence alignment using clustalw showed that the identity of vam and the watermarked vam sequence is %. the mutagenized base pairs are localized in the snare domain, which is thought to be essential for the homotypic fusion. the integrated watermark did not influence the function of vam and the resulting phenotype of the cg cells transformed with prs vam7-tb show no significant differences compared to the cg cells transformed with prs vam the vacuolar morphology in both transformed strains was similar compared to the wild type strain. the number of vacuoles was slightly increased compared to the wild type strain. as shown in figure cg cells transformed with prs vam and cg cells transformed with prs vam7-tb display no significant differences, which points to the fact that the insertion of the watermark has no influence to the resulting protein. in contrast to strain cg which is not able to sporulate most likely due to the lack of a functionally active vam in cg cells, % ± of the prs vam and % ± of the prs vam7-tb transformed yeast cells formed spores. the spores of the transformed cg cells displayed the normal phenotype of cg spores. also in these experiments the insertion of the watermark did not result in an altered phenotype, comparing cg cells transformed with prs vam and cg cells transformed with prs vam7-tb. cg cells and the cg cells transformed with prs vam or prs vam7-tb, respectively, displayed a higher division rate compared to cg cells. the division rate is calculated by odt9odt0=a with measuring points ti, i =.., in addition there are no significant differences comparing the division rates of the cg cells transformed with prs vam and the the cg cells transformed with prs vam7-tb. our aim was to prove that the insertion of the watermark'tb' into the prs dna does not influence the expression of a functional vam gene. by testing the morphology and several growth parameters we demonstrate that the cells containing prs or prs plus the watermark tb are indistinguishable from each other with respect to the parameters verified. the vam negative and untransformed wild type cells were included in the experiments as controls. previously reported in silico studies using the ypt genes of saccharomyces cerevisiae demonstrated that using watermarks in coding regions did not influence the resulting protein. in this paper we show for the first time that the in silico results could be confirmed in vivo by analyzing living yeast cells.  to our knowledge the insertion of watermarks into eukaryotic cells has not been reported so far. only storage of information in bacteria has been published. as prokaryotic and eukaryotic cells show very different complexities e. g. on the levels of transcription, translation or compartmentalization it is very important to test and prove the application of dna watermarks into coding regions of living eukaryotic cells. and this to our knowledge was successfully done for the first time in the experiments which we report on in this manuscript. our dna-crypt algorithm clearly represents advances compared to the algorithms reported by wong et al. or arita and ohashi. it permits the use of several mutation detection and correction codes, like the hamming-code or the wdh-code and binary encryption algorithms like aes, blowfish or rsa. further it provides the use of one-time pads. additionally the dna-crypt algorithm allows an increased amount of information per base and further has an integrated fuzzy controller that recommends whether to use a specific correction code or not. although, for reasons of economy, we inserted only'tb' into the vam gene more than two letters could be integrated without any effect to the resulting protein, because dna-crypt only produces silent mutations. to introduce longer watermark sequences more expensive synthesis of oligonucleotides and extended mutagenesis procedures would have to be performed. nethertheless the resulting observations would be the same based on the fact that dna-crypt only produces silent mutations. our experiments using the vam gene are a proof of concept for introducing dna watermarks into coding regions and can be generalized for other proteins. the use of dna watermarks in non coding or regulatory sequences will be subject to further examinations. 
the cg cells are unable to sporulate and in addition display an abnormal vacuolar morphology. by analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting vam protein was functionally active. from our experiments we conclude that the dna watermarks produced by dna-crypt do not influence the translation from mrna into protein. transformation of cg with prs vam leads to a phenotype very similar to the wildtype yeast strain cg the integrated watermark did not influence the function of vam and the resulting phenotype of the cg cells transformed with prs vam7-tb shows no significant differences compared to the cg cells transformed with prs vam for our investigations we used a mutant saccharomyces cerevisiae strain, called cg which has an amber mutation within the vam sequence. in silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the dna-crypt algorithm.
 dna-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms protected by patents. in silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the dna-crypt algorithm.  we integrated an authenticating watermark in the vam sequence. for our investigations we used a mutant saccharomyces cerevisiae strain, called cg which has an amber mutation within the vam sequence. the cg cells are unable to sporulate and in addition display an abnormal vacuolar morphology. transformation of cg with prs vam leads to a phenotype very similar to the wildtype yeast strain cg the integrated watermark did not influence the function of vam and the resulting phenotype of the cg cells transformed with prs vam7-tb shows no significant differences compared to the cg cells transformed with prs vam  from our experiments we conclude that the dna watermarks produced by dna-crypt do not influence the translation from mrna into protein. by analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting vam protein was functionally active. 