Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RBI) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 λ phage clones spanning the whole RBI gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q 14. Normal interphase nuclei showed two RBI signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RBI gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RBI gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RBI locus. This indicates that LOH at the RBI locus in breast cancer cells probably involves mechanisms other than physical deletion.
ASJC Scopus subject areas
- Molecular Biology