Figure 4

Methylation Status of Human Intronless GLTP Gene. Vertical bars show location of CpG dinucleotides. Bst UI restriction sites, consisting of adjacent CpG dinucleotides, are shown by scissors. Individual clones are shown by horizontal rows of circles. Filled circles represent methylated CpG dinucleotides; unfilled circles represent nonmethylated CpG dinucleotides. Primers for RT-PCR (255 base product) are indicated by dotted arrows. The ATG start codon represents nucleotides +1, +2, and +3. B. & C. Combined bisulfite restriction analysis (COBRA) of intronless GLTP gene methylation status. B. Agarose gel electrophoresis patterns obtained before (Lane 1) and after (Lane 2) Bst UI restriction digestion of bisulfite-treated PCR products for intronless GLTP gene (GLTPi) in human blood cell genomic DNA. C. COBRA analysis of intronless GLTP methylation status in different cell types. Lane 1 = pooled PCR products before Bst UI restriction digestion; Lanes 2–9 shows action of Bst UI restriction digestion. Lane 2 = glioma cells; Lane 3 = human skin fibroblasts; Lane 4 = human HBL100 breast cancer cells; Lane 5 = Gaucher cells; Lane 6 = human T47D breast cancer cells; Lane 7 = human IMR32 neuroblastoma cells; Lane 8 = human HTB126 breast cancer cells; Lane 9 = human Caov3 ovarian cancer cells. Because of the absence of Bst UI restriction sites in amplified PCR products of the CpG island of the 5-exon/4-intron region, COBRA analysis could be used to verify the absence of methylation detected by direct sequencing and served only as a negative control for Bst UI action shown Panel C. Std = molecular weight standards.