High-resolution physical map and transcript identification of a prostate cancer deletion interval on 8p22

Abstract

A genomic interval of approximately 1-1.5 Mb centered at the MSR marker on 8p22 has emerged as a possible site for a tumor suppressor gene, based on high rates of allele loss and the presence of a homozygous deletion found in metastatic prostate cancer. The objective of this study was to prepare a bacterial contig of this interval, integrate the contig with radiation hybrid (RH) databases, and use these resources to identify transcription units that might represent the candidate tumor suppressor genes. Here we present a complete bacterial contig across the interval, which was assembled using 22 published and 17 newly originated STSs. The physical map provides twofold or greater coverage over much of the interval, including 17 BACs, 15 P1s, 2 cosmids, and 1 PAC clone. The position of the selected markers across the interval in relation to the other markers on the larger chromosomal scale was confirmed by RH mapping using the Stanford G3 RH panel. Transcribed units within the deletion region were identified by exon amplification, searching of the Human Transcript Map, placement of unmapped expressed sequence tags (ESTs) from the Radiation Hybrid Database (RHdb), and from other published sources, resulting in the isolation of six unique expressed sequences. The transcript map of the deletion interval now includes two known genes (MSR and N33) and six novel ESTs.

Figure 1

Physical and transcript map of the 8p22 homozygous deletion interval. (A) Extent of the target interval (Bova et al. 1996; Levy et al. 1999). (B) Selected YAC clones that cover the deletion interval and their approximate extent (Bookstein et al. 1994). The numbers refer to the CEPH database YAC name; the approximate sizes of YAC clones are given in parenthesis. (C) Long-range restriction map of the YAC clones encompassing the interval (adapted from Bova et al. 1996). (D) Two-point distances between the indicated markers in cR based on the analysis with the Stanford G3 RH panel. (E) Minimal-tiling path contig of the deletion interval. Only selected markers are shown for simplicity. Those shown in boldface type have also been used for RH analysis. Vertical gray lines connect markers to the bacterial clones with which they tested positive. (Prefix P) PI clones; (PA) PAC clones; (B) BAC clones; (C) cosmid clones. (F) Location of known genes and partial cDNAs described in Tables 3 and 4. Horizontal bars indicate the clones to which transcripts have been localized.

Figure 2

Complete list of the isolated bacterial clones and their STS content. Marker names are listed across the top row. Markers who order is unresolved in relation to other markers that map to the same clone(s) are indicated by horizontal solid bars at top. Clone names are listed in the left column in order from telomere (top) to centromere (bottom). (Prefix P) PI clones; (PA) PAC clones; (B) BAC clones; (C) cosmid clones. STS content of each clone is indicated by a solid box next to the clone name.

Figure 3

Representative pictures of RT–PCR analysis on selected epithelial cancer cell lines. RT–PCR was performed on total cellular RNA. First-strand cDNA was synthesized using oligo(dT); PCR primers were designed from each partial cDNA clone. The lanes are as indicated. Control reactions without reverse transcriptase and reactions containing water were performed for every sample.