Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.
We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 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 ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.
Akiyama
H
H
Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto 606-8507, Japan. akiy@kuhp.kyoto-u.ac.jp
Hiraki
Y
Y
Noda
M
M
Shigeno
C
C
Ito
H
H
Nakamura
T
T
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001853:Bone Marrow / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D015723:Gene Library; D011905:Genes, ras; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D019151:Peptide Library; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection
D019151:Peptide Library; D012333:RNA, Messenger