FIG. 1.

WRN promoter region and structures of plasmids used for promoter assays. (A) Orientation of WRN in the P1/PAC contig map. Solid lines and numbers represent P1/PAC DNAs that form the physical map. The approximate positions of WRN and the STS marker D8S2162 are shown. The transcriptional direction of WRN is indicated by 5′ and 3′. Tel., telomere; Cent., centromere. (B) The 2,776-bp DNA fragment generated from PAC 12339 DNA was placed at the BglII site of the pGL3-Basic vector upstream of the firefly luciferase gene to form pGL3/A, which was used as a reporter gene in the promoter assay. A series of WRN-Luc plasmids containing the sequentially 5′-deleted human WRN (hWRN) promoter regions (pGL3/B to -R) were prepared from pGL3/A. The structures of Rb and p53 expression plasmids (pCMV1-Rb, pC53-SN3, and pC53-249) are shown. CMV, cytomegalovirus. (C) The WRN transcription start sites are indicated by asterisks. The most upstream nucleotide position is assumed to be position +1. The cis-acting Sp1 element binding sites and an RCE motif are boxed and underlined, respectively. (D) Results of S1 nuclease protection assay. The ladder (lanes A, C, G, and T) represents the sequence of pGEM3Zf(+) DNA recognized by the M13 forward primer (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′), and base 102 is shown by an arrowhead. Lane 1, S1 mapping with 10⁶ cpm of ³²P-labeled probe DNA for K562 cell RNA that contains WRN mRNA; lane 2, control experiment using yeast tRNA; lane 3, S1 nuclease-undigested 5′ ³²P-labeled 245-bp DNA (10⁴ cpm) that encompasses the potential WRN promoter region from −143 to +102, heat denatured prior to electrophoresis. The band remaining in the upper part of the gel indicates the S1 nuclease-resistant duplex DNA formed by annealing of complementary DNAs during hybridization. The band migrating with base 102 (lane 1) corresponds to the most upstream nucleotide from the ³²P-labeled 5′ G (from the complementary strand to the 3′-5′ CCG indicated by an arrow in panel C) and is designated the +1 position of the WRN mRNA.