A dual-color FISH framework map for the characterization of the Sai1 tumor suppression region on rat chromosome 5.

The analysis of cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts has previously demonstrated the critical role of a deletion in rat chromosome 5 (RNO5) that was related to an anchorage independent phenotype. Those hybrids that were anchorage independent displayed loss of the entire RNO5 or an interstitial deletion in RNO5. These findings suggested that a putative tumor suppressor gene, Sai1 (suppression of anchorage independence 1), was located within the deleted region. To explore the molecular basis of the tumor suppressor activity of the Sai1 region, we analyzed the RNO5q23-q36 region with several genes and microsatellite markers that could be assigned to the region, as well as with new markers derived by representational difference analysis (RDA) or by microdissection. Dual-color FISH was used to construct a detailed physical map of the entire RNO5. These new data can be used to connect the physical and linkage maps in the rat, as well as to identify the details of the comparative map with other mammalian species including humans and mice. Using as FISH reagents genomic YAC, P1, or phage lambda clones corresponding to RNO5 markers, the order and unique positions of 18 markers could be established. The map provided a framework for the detailed characterization of the deletion found in anchorage independent hybrids. All markers within the bands RNO5q31.3-q35 were shown to be lost, including known cancer-related genes such as Ifna (5q32), Cdkn2a, -b (5q32), Jun (5q34), and Cdkn2c (5q35). However, the aberration in the deletion chromosome turned out to be more complex than originally thought in that we detected the presence of a paracentric inversion in addition to a deletion. The inversion led to the juxtaposition of the gene markers Tal2 (5q24.1) and Cd30lg (5q24.3). The framework map will provide the basis for the detailed physical YAC clone contig mapping of this region, and facilitate the identification and characterization of the Sai1 locus.

Isolation of DNA markers for the rat Sai 1 gene for suppression of anchorage independence by using representational difference analysis.

We have applied the representational difference analysis (RDA) to isolate genetic markers for a deletion on the rat chromosome RNO5q22-33. This deletion occurred in anchorage independent sublines of a normal rat fibroblast x mouse hepatoma cell hybrid (BS181) (Islam 1989). Normal rat tissue DNA provided the "tester" and the BS181 hybrid DNA the "driver" in the RDA hybridization/selection reactions. Out of twelve RDA derived DNA sequences that were analyzed in detail using a rat X mouse cell hybrid panel for chromosome mapping, nine (75%) were found to represent RNO5 deletions, whereas the other three were new RFLPs mapping to other chromosomes. In two cases, the RDA sequences were also analyzed by fluorescence in situ hybridization (FISH) and found to give distinct signals in the RNOq22-33 region. This result emphasizes teh significance of the previous cytogenetic analysis of this hybrid, which indicated the presence of a gene for the suppression of anchorage independence, Sai 1, in this deletion region. The RDA derived sequences isolated by this work will provide a valuable source of new genetic markers for the further detailed analysis of the Sai 1 deletion region.