Genomic structure ofp57^KIP2 gene and location of primers used in this study.

Short vertical bars on bottom line show sites of CpG(1-46) analyzed by bisulfite sequencing; open rectangle, noncoding region ofp57^KIP2 gene transcripts; shaded rectangle, coding region ofp57^KIP2 gene transcripts; closed circle, putative transcriptional factor binding site (Hatada and Mukai22); S1-S5, primer sets for PCR–single-strand conformation polymorphism (PCR-SSCP) (Hatada and Mukai22); KIP2-1 and KIP2-2, primer set of RT-PCR (see “Materials and methods”). Methylation-specific PCR (MSP) with the use of primer sets specific for methylated DNA (MSP-M), MSP with the use of primer sets specific for unmethylated DNA (MSP-U), and MSP-A indicate primers for MSP analysis (see “Materials and methods”); MS1, MS2, MS3, and MS4, primers for bisulfite sequencing analysis (see “Materials and methods”). Locations of these primers (in relation to the transcriptional start site ofp57^KIP2 gene) are as follows: MSP-M, bp −282 to −263; MSP-U, bp −289 to −263; MSP-A, bp −41 to −20; MS1, bp −460 to −441; MS2, bp +75 to +91; MS3, bp −418 to −401; MS4, bp −148 to −125.