A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme. The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H – HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with32P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 – 4N, 4T, lanes 3, 4 – 5N, 5T, lanes 5,6 – 7N, 7T respectively. Case numbers correspond to that indicated in table 1. The arrows show the positions of 28S and 18S RNA. C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 680 bp32P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 – DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 – DNA digested by HpaII; lanes 1, 2 – 9N; lanes 3, 4 – 9T; lanes 5,6 – 4N; lanes 7, 8 – 4T; lanes 9, 10 – 7N; lanes 11,12 – 7T; lanes 13,14 – SiHa; lanes 15, 16 – HeLa. Case numbers correspond to that indicated in table 1. D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L). All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table 1; mr – molecular weight marker.