Molecular cloning and biological activity of a novel Ha-ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrowby differential display using clonal mouse EC cells, ATDC5

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differentia l display between two mouse cell lines: embryonic fibroblast C3HIOT1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-CI consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATD C5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 b one marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-CI mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mi ce. By in situ hybridization, A-C1 gene expression was localized in hippoca mpus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as web as perinuclear region of the cells. Trans fection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell Line caused increa se in the number of flat colonies and inhibition of cell growth. Our data i ndicate that A-C1 is expressed in some specific tissues in vivo and modulat es Ba-ras-mediated signaling pathway.