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

Abstract

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.