Cloning of differentially expressed genes in highly and low metastatic ratosteosarcomas by a modified cDNA-AFLP method

To identify differentially expressed genes between highly and low metastati c rat transplantable osteosarcomas, we applied a modified AFLP (amplified f ragment length polymorphisms) method for cDNA subtraction. The specific poi nt of our modification is selective amplification using suppression PCR tec hnique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducib ility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology wi th known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement mem brane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologu e of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effecters of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and prolife rative markers may be important for metastasis of osteosarcomas. (C) 1999 A cademic Press.