Introduction to the Workshop
URLs Provided by Attendees
- Ethical, Legal, and Social Issues
The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
IDENTIFICATION AND MOLECULAR CLONING OF A 20q13.2 AMPLICON IN BREAST CARCINOMA
Colin Collins, Jeff Froula, Minna Tanner, Jan-Fang Cheng, David Kowbel, Farideh Shadravan, Chris Martin, Michael Palazollo, Mary Hintz, Ulli Weier, Wen-Lin Kuo, Olli Kallioniemi, Jeff Gingrich, Johanna Rommens, Dan Pinkel[1,2] and Joe Gray[1,2]
Lawrence Berkeley Laboratory, Berkeley, California; University of California, San Francisco, California; Tampere University Hospital, Tampere, Finland; Lawrence Livermore National Laboratory, Livermore, California; Hospital for Sick Children, Toronto, Ontario.
Comparative genomic hybridization (CGH) has identified a previously undescribed region of increased copy number involving chromosome band 20q13.1-13.2 in 15-20% of primary breast carcinomas. The application of FISH to the study of tumor interphase nuclei using 33 locus specific cosmid and P1 probes distributed along chromosome 20 revealed amplification of band 20q13.2 in 35% of breast cancer cell lines and 8% of primary tumors, and localized the amplification event to the approximately 2 Mb interval defined by (Flpter 0.80-0.84.). This excludes all known genes in the region as candidates for the putative oncogene(s). It is hypothesized that selection for the overexpression of a novel oncogene(s) is driving the observed increase in copy number.
A 3 Mb 12 member YAC contig has been assembled that spans the region of increased copy number. Conversion of the YAC contig to a P1 contig is proceeding by three approaches. In the first approach, interAlu PCR reactions are performed on individual YACs and sufficient products sequenced to create 5-10 STSs per YAC clone. The DuPont P1 library is then screened for these STSs by the PCR. In the second approach, the entire interAlu PCR product is used as a hybridization probe against a gridded array of the DuPont P1 library. Finally, P1 ends are sequenced and STSs created for P1 library screening. These complementary approaches have yielded 52 P1 clones forming 12 contigs. STS content mapping, P1 Southern hybridizations, and P1 fingerprinting are being employed to merge the P1 clones into a single contig. All 52 P1s are being hybridized to tumor derived cell lines and a panel of primary breast tumors using interphase FISH to precisely define the minimum common region of increased copy number.
The direct selection of cDNAs and exon trapping are being performed to identify genes encoded within the 20q13.2 amplicon. In collaboration with the Human Genome Center at the Lawrence Berkeley Laboratory we have initiated the directed sequencing of a P1 contig localized at the postulated core of the 20q13.2 amplicon. It is expected that the combination of large scale directed sequencing, exon trapping and direct selection will culminate in the isolation of the hypothesized oncogene(s) and contribute to the development of novel molecular diagnostic and therapeutic approaches for the management of breast cancer.
This work was supported by grants from US DOE contract DEAC0376SF00098, USPHS grants CA44768, CA45919, CA52807 and Imagenetics.