Identification of transcriptional initiation sites of the MEL1S gene, and DNA methylation. (A) The 5′-RACE was performed with cDNA synthesized using mRNA from an ATL cell line, ATL-55T. Primers in exon 6 were used to amplify cDNA. Two major bands were detected. (B) Schema of transcriptional initiation sites of the MEL1S gene in ATL cells. PCR products of 5′-RACE were cloned into plasmid DNA and then their sequences were determined. The majority of MEL1S transcripts initiated from upstream of exon 6 (633 bp [1] and 410 bp [2] upstream from exon 6 as shown in Figure 4B). Transcription started 477 bp upstream of the exon 4, which contained putative exon (▦; 154 bp [3]). From these data, 2 major initiation sites were identified as shown in 1 and 2. (C) The methylation status surrounding the transcriptional initiation sites of MEL1S gene and promoter region of MEL1 gene. Genomic DNA was treated by sodium bisulfite and amplified by specific primers. The methylation status was determined by sequencing of these PCR products. The schema shows the structure of the MEL1/1S gene and transcriptional initiation sites of the MEL1S gene are shown by arrows (1, 2, and as described for panel B). Methylation of CpG sites surrounding the transcriptional initiation sites of MEL1S is shown (•, methylated; ○, unmethylated).