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

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.     

Evaluation of mitochondrial function by measuring the heat production in state 3 and state 4 respiration

Using a microcalorimetric method, we have measured the heat production in states 3 and 4 respiration of a mitochondrial preparation from rat heart ventricle. Adenosine triphosphate production in state 3 respiration was also determined for the same preparation after heat production was measured. In Tris-buffered solution with pyruvate and malate used as substrates, the total heat production in states 3 and 4 respiration for a 10-min reaction period were about 709 and 207 mJ/mg of mitochondrial protein, respectively. 2,4-Dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation, increased the heat production in both states 3 and 4 respiration. On the other hand, antimycin A1, an inhibitor of the electron transport system, decreased the heat production in both states. When ventricle mitochondrial dysfunction was induced by the in vivo administration of a high dose of isoproterenol, heat production was increased in state 4 respiration. These results indicate that the calorimetric method can be used to evaluate the mitochondrial function.     

Somatic-cell mutation induced by UVA and monochromatic UV radiation in repair-proficient and -deficient Drosophila melanogaster

Near-ultraviolet light (UVA: 320-400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair-deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 +/- 1.5 spots/wing/kJ vs 0.3 +/- 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 +/- 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 +/- 2.9 x 10(-3) vs 3.1 +/- 1.1 x 10(-3) spots/wing/kJ; at 340 nm, 3.5 +/- 0.9 x 10(-3) vs 2.0 +/- 0.7 x 10(-3). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.     

DNA methylation analysis: speedup of bisulfite-mediated deamination of cytosine in the genomic sequencing procedure

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The deamination is usually performed using sodium bisulfite solutions of 3–5 M concentration with an incubation period of 12–16 hr at 50 °C. We demonstrate here that this deamination can be speeded up significantly. We prepared a solution of 10 M bisulfite concentration of pH 5.4 and used it to treat DNA at temperatures up to 90 °C. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at 90 °C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer a significantly improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.     

The Breit Equation

Contrary to the conventional view, the Breit equation can be solved. This revised version was published online in August 2006 with corrections to the Cover Date.