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BIOINFORMATICS APPLICATIONS NOTE Vol. 24 no. 17 2008, pages 1961–1962 doi:10.1093/bioinformatics/btn342 Genetics and population analysis Watermarking sexually reproducing diploid organisms Dominik Heider, Daniel Kessler and Angelika Barnekow∗ Department of Experimental Tumorbiology, University of Muenster, Badestrasse 9, 48149 Muenster, Germany Received on April 30, 2008; revised and accepted on July 2, 2008 Advance Access publication July 3, 2008 Associate Editor: Martin Bishop ABSTRACT Summary: DNA watermarks are used for hiding messages or for authenticating genetically modified organisms. Recently, we presented an algorithm called DNA-Crypt for generating DNAbased watermarks that can be integrated into the genome by using the characteristics of the degenerative genetic code. DNA-Crypt generates the watermark by replacing single bases and thus creating synonymous codons that encrypt the hidden information. Mutations within the integrated DNA sequence can be corrected using several mutation correction codes, to keep the hidden information intact. This method has successfully been tested in asexually replicating organisms like bacteria or yeast, where the watermark is duplicated with every cell division. It has been shown that DNA watermarks produced by DNA-Crypt do not influence the transcription or translation of a protein. In sexually reproducing diploid organisms, additional problems can occur, e.g. recombination events can destroy hidden information. Using population predictions as well as statistical analyses we identified a coupled Y-chromosomal/mitochondrial DNA watermarking procedure as the most appropriate for diploid organisms. We developed a mitochondria adapted version of DNA-Crypt, which is called Project Mito that can be used in combination with the original program. Availability: http://www.uni-muenster.de/Biologie.NeuroVer/ Tumorbiologie/DNA-Crypt/index.html Contact: [email protected] Password: WWUTB Requirements: Java 5.0 or higher Supplementary information: Supplementary data are available at Bioinformatics online. 1 INTRODUCTION DNA watermarks have recently been used for authentication or identification of genetically modified organisms (GMOs) (Heider and Barnekow, 2007). It has been shown in silico, in vitro and in vivo that these watermarks do not affect the translation of mRNA and that the resulting protein is functionally intact (Heider and Barnekow, 2007, 2008). Watermarks in asexually reproducing organisms are passed on with every cycle of cell division. The only mechanism by which a watermark can be lost is in the case of mutation events, which can be avoided using mutation correction codes (Heider and Barnekow, 2007). ∗ To whom correspondence should be addressed. Using sexually replicating organisms instead of asexually dividing individuals new problems evolve for DNA watermarking. Diploid chromosome sets represent a new challenge. In addition, recombination and crossover events can destroy integrated watermarks. 2 APPROACH Population predictions revealed that coincidental mating with a genetic drift of non-watermarked alleles leads to a reduction or loss of watermarks. An autosomal transmitted watermark can be disrupted from one generation to the next by recombination events. Y-chromosomal linked heredity leads to a male watermarked line, female offspring cannot be watermarked using the Y-chromsome. In contrast to the Y-chromosomal heredity, a mitochondrial transmissed watermark leads to a female watermarked line, as mammalian mitochondrial DNA (mtDNA) is mainly maternally and only 0.01—0.1% paternally inherited (Schwartz and Vissing, 2003). To combine the positive effects of all transmission modes and to suppress the negative impacts it is necessary to link different watermark constellations. An X-/Y-chromosomal linked inheritance leads to a reduction of watermark containing individuals starting with the third generation (Fig. 1). A Y-chromosomal linked heredity combined with a mitochondrial transmission would be the best alternative to preserve the watermark (Fig. 1). Both the X-/Y-chromosomal linked transmission of watermarks and the Y-/mitochondrial mode of transmission result in a decreasing number of watermarked individuals from one generation to the next regarding the whole population (limn→∞ = 0). The survival of the watermark using an X-/Y-chromosomal linked procedure depends on the 50% probability to transmit the watermarked X chromosome from mother to son. In the worst case constellation (with respect to the maintenance of the watermark), the watermarked X chromosome is never inherited and therefore, the worst case of an X-/Y-chromosomal linked watermark procedure is the minimum number (R) of watermarked individuals within a generation n: 1 Rw (n) = n (1) 2 and in the whole population (G) n+10 ,n ≥ 5 Gw (n) = n k k=1 2 (2) Compared to the X-/Y-chromosomal mode of transmission, the Y-/mitochondrial-based transmission of watermarks yields a partial stabilization of a female and a male watermarked line, because © The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1961 D.Heider et al. Fig. 1. Transmission of watermarks. (A) X-/Y-chromosomal transmitted watermark; (B) Y-chromosomal/mitochondrial transmitted watermark. Watermarked chromosomes and mitochondria (*) are bold and underlined. a male individuum always inherits the watermarked Y chromosome from his father and every offspring always inherits the watermarked mt DNA from its mother, which leads to the following number (R) of watermarked individuals in generation n: 3 R(n) = n 2 and (3) 4+3×(n−2) ,n ≥ 5 (4) n k k=1 2 in the whole population (G). The detailed calculations of the formulas 1 to 4 can be found in the Supplementary material. Thus we recommend the use of Y-/mitochondrial inheritence for more stably watermarking sexually reproducing organisms. As mtDNA differs from nuclear DNA in some aspects, watermarking procedures have to be adjusted to these conditions. Today, most of the genetic information needed for mitochondrial function is coded within the nucleus and the gene products are transported into mitochondria (Gabaldon and Huynen, 2004; Kessler et al., 2007). The mtDNA itself still encodes some components of the mitochondrial translation machinery (rRNA and tRNA) as well as structural components of the oxidative chain (Garesse and Vallejo, 2001). The tRNA molecules are necessary to use a mitochondriaspecific genetic code that slightly differs from the universal code. In addition, policistronic regions offer a special challenge. The mtDNA displays a higher mutation rate compared to cellular genes, which emphasizes the necessity for a mutation correction code (Heider and Barnekow, 2007). In an in silico analysis we identified a region within the human mtDNA, which is suitable for inserting a watermark. This region G(n) = 1962 within the Cytochrome c oxidase subunit I gene spans the range from 5904–7445 bp containing no overlapping gene regions. The watermarking algorithms had to be modified to meet the special requirements of mtDNA. Therefore, we developed a program called Project Mito for creating mitochondrial watermarks. Project Mito is derived from the original DNA-Crypt and can be used in combination with the DNA-Crypt algorithm. The binary file, which is thought to be encrypted into DNA is first modified by a mutation correction code, the Hamming-code, to correct mutations within the DNA sequences. A header, containing the length of the original file, is produced and linked to the modified binary sequence. The composition of the header and the file is translated into a DNA sequence, which is embedded into the mtDNA using synonymous codons as described (Heider and Barnekow, 2007). To extract the information within the DNA watermark, Project Mito reads out the first synonymous codons within the target gene and reconstructs the binary header, which notifies Project Mito about the length of the intergrated information. Possible mutations occuring in the DNA sequences are corrected by Project Mito itself and the DNA sequence is translated into the binary file. Users should follow the instructions of the manual, which is provided on the project’s homepage. Using Project Mito we were able to design a first potential watermark sequence that could be inserted into the above described region within human mtDNA. Analyses of the Cytochrome c oxidase subunit I gene using Project Mito shows that the gene can carry a maximum information of 60 bytes. In our experiments the integrated watermark sequence contains the message ‘Project Mito’. A pairwise sequence alignment displays an identity of about 95% between the watermark sequence and the original sequence. These differences in the DNA sequence do not alter the resulting protein as described. Taken together we presented and tested in silico a watermarking procedure that can be used for authentication of genetically modified mammalian cells or even in animal breeding processes based on a Y-/mitochondrial transmission. ACKNOWLEDGEMENTS The authors thank Daniel Tenbrinck (Institute of Computer Science, University of Muenster) for verifying the statistical calculations. This work is part of the PhD thesis of D.H. Conflict of Interest: none declared. REFERENCES Gabaldon,T. and Huynen,M.A. (2004) Shaping the mitochondrial proteome. Biochim. Biophys. Acta, 1659, 212–220. Garesse,R. and Vallejo,C.G. (2001) Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes. Gene, 263, 1–16. Heider,D. and Barnekow,A. (2007) DNA-based watermarks using the DNA-crypt algorithm. BMC Bioinformatics, 8, 176. Heider,D. and Barnekow,A. (2008) DNA watermarks: a proof of concept. BMC Mol. Biol., 9, 40. Kessler,D. et al. (2007) The DNA binding parvulin par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in hominidae. BMC Biol., 5, 37. Schwartz,M. and Vissing,J. (2003) New patterns of inheritance in mitochondrial disease Biochem. Biophys. Res. Commun., 310, 247–251.