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Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account.

Hayatsu H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2008)

Bottom Line: At the same time, Shapiro and his coworkers in New York University found the same reaction independently.We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly.The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan. hayatsuh@cc.okayama-u.ac.jp

ABSTRACT
Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

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Two diastereomeric thymine-bisulfite adducts formed in the deamination of 5-methylcytosine.
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f6-321: Two diastereomeric thymine-bisulfite adducts formed in the deamination of 5-methylcytosine.

Mentions: We observed that 5-methylcytosine can be deaminated by bisulfite, but much more slowly than cytosine.9) Further studies showed that the rate of the deamination is nearly two orders of magnitude lower than that for cytosine.12) Moreover, it was revealed that the deaminated products, thymine-bisulfite adducts are a mixture of two diastereomers.13) The structures of these isomers are shown in Fig. 6. They were produced in approximately 1 to 1 ratio. The trans isomer is the same adduct as that formed in the treatment of thymine with bisulfite (trans, regarding the –SO3− at position 6 and the –H at position 5). This trans isomer readily generates thymine on treatment with alkali. In contrast, the other one, cis adduct, is quite stable. It can be converted to thymine only with a harsh alkaline treatment. It had been common knowledge that cis–elimination is difficult to occur.14) Ehrlich and coworkers reported in 1980 that bisulfite-mediated deamination of 5-methylcytosine in DNA in fact produced such alkali-resistant products.15)


Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account.

Hayatsu H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2008)

Two diastereomeric thymine-bisulfite adducts formed in the deamination of 5-methylcytosine.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722019&req=5

f6-321: Two diastereomeric thymine-bisulfite adducts formed in the deamination of 5-methylcytosine.
Mentions: We observed that 5-methylcytosine can be deaminated by bisulfite, but much more slowly than cytosine.9) Further studies showed that the rate of the deamination is nearly two orders of magnitude lower than that for cytosine.12) Moreover, it was revealed that the deaminated products, thymine-bisulfite adducts are a mixture of two diastereomers.13) The structures of these isomers are shown in Fig. 6. They were produced in approximately 1 to 1 ratio. The trans isomer is the same adduct as that formed in the treatment of thymine with bisulfite (trans, regarding the –SO3− at position 6 and the –H at position 5). This trans isomer readily generates thymine on treatment with alkali. In contrast, the other one, cis adduct, is quite stable. It can be converted to thymine only with a harsh alkaline treatment. It had been common knowledge that cis–elimination is difficult to occur.14) Ehrlich and coworkers reported in 1980 that bisulfite-mediated deamination of 5-methylcytosine in DNA in fact produced such alkali-resistant products.15)

Bottom Line: At the same time, Shapiro and his coworkers in New York University found the same reaction independently.We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly.The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan. hayatsuh@cc.okayama-u.ac.jp

ABSTRACT
Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

Show MeSH