Mti

18S rRNA

Fig. 6. Northern blot analysis of MT-I mRNA in lymphosarcoma cells and in Morris hepatoma before and after 5-azaC treatment. RNA (30 /zg) isolated from cells or tissues is analyzed by Northern blotting first with random-primed, 32P-labeled mouse MT-I cDNA and subsequently with rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. [From S. Majumder, K. Ghoshal, Z. Li, Y. Bo, and S. T. Jacob, Oncogene 18,6287 (1999); and K. Ghoshal, S. Majumder, Z. Li, X. Dong, and S. T. Jacob, J. Biol. Chem. 275, 539 (2000).]

Chromosomal DNA was isolated from mouse thymus and PI798 cells (with or without 5-azaC treatment for 72 hr), and then treated with bisulfite reagent. MT-I promoter (from —526 bp to —2 bp) is amplified with gene-specific primers. PCR-amplified DNAs from all three samples are fully cleaved by Apol and 7sp509I, respectively, indicating complete bisulfite conversion (Fig. 7A). To detect the presence of CpG sites, if any, after bisulfite treatment, the amplified DNA is digested

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