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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.

Show MeSH
Permanganate oxidation of 4-thiouridine to form its sulfonate derivative.
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Related In: Results  -  Collection


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f2-321: Permanganate oxidation of 4-thiouridine to form its sulfonate derivative.

Mentions: The major training I received in the undergraduate- and the 2-year graduate-courses, 1955–1958, in the University of Tokyo, was organic chemistry, under the guidance of the great chemist, Professor E. Ochiai (1898–1974), whose expertise was synthesis and reactions of aromatic amine oxides. He dictated to students that a chemist may read literatures but should not be led by them. I happily complied with this instruction and spent most of my time doing experimental bench-work. This life-style of mine has persisted many decades until today. My doctoral work under the supervision of Professor T. Ukita (1915–1972) was chemical synthesis of nucleotides, and then I did post-doctoral work in Professor H. G. Khorana’s laboratory in the University of Wisconsin, 1964–1967, synthesizing oligonucleotides. Coming back to Tokyo, I began searching for reagents that can modify DNA bases in aqueous solutions under mild conditions. Soon I found that potassium permanganate, a commonly used oxidizing agent, is capable of modifying thymine selectively among major bases.5) Along with that work, a finding was made; i.e., a fast reaction of 4-thiouridine, a minor nucleoside in transfer RNA, with permanganate, forming its 4-sulfonate6) (Fig. 2). It became necessary to measure the progress of this oxidation, and for that purpose, the permanganate in the reaction mixture had to be degraded by adding a reducing agent. The reducing agent I chose was sodium bisulfite. Then, an unexpected phenomenon was observed.


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)

Permanganate oxidation of 4-thiouridine to form its sulfonate derivative.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-321: Permanganate oxidation of 4-thiouridine to form its sulfonate derivative.
Mentions: The major training I received in the undergraduate- and the 2-year graduate-courses, 1955–1958, in the University of Tokyo, was organic chemistry, under the guidance of the great chemist, Professor E. Ochiai (1898–1974), whose expertise was synthesis and reactions of aromatic amine oxides. He dictated to students that a chemist may read literatures but should not be led by them. I happily complied with this instruction and spent most of my time doing experimental bench-work. This life-style of mine has persisted many decades until today. My doctoral work under the supervision of Professor T. Ukita (1915–1972) was chemical synthesis of nucleotides, and then I did post-doctoral work in Professor H. G. Khorana’s laboratory in the University of Wisconsin, 1964–1967, synthesizing oligonucleotides. Coming back to Tokyo, I began searching for reagents that can modify DNA bases in aqueous solutions under mild conditions. Soon I found that potassium permanganate, a commonly used oxidizing agent, is capable of modifying thymine selectively among major bases.5) Along with that work, a finding was made; i.e., a fast reaction of 4-thiouridine, a minor nucleoside in transfer RNA, with permanganate, forming its 4-sulfonate6) (Fig. 2). It became necessary to measure the progress of this oxidation, and for that purpose, the permanganate in the reaction mixture had to be degraded by adding a reducing agent. The reducing agent I chose was sodium bisulfite. Then, an unexpected phenomenon was observed.

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