MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA SHOWING NEGATIVE REGULATION IN V-SRC-TRANSFORMED 3Y1 RAT FIBROBLASTS

A differential screening procedure was used to isolate genes that are specifically down-regulated in transformed cells. After screening a cDNA library derived from normal rat fibroblast (3Y1) cells, we obtained several independent cDNAs whose mRNA level was substantially lower in 3Y1 cells transformed with Rous sarcoma virus than in untransformed 3Y1 cells. Among them, NO3 cDNA has been characterized extensively. NO3 mRNA was also absent from v-mos- or simian virus 40-transformed 3Y1 cells but was present in v-Ha-ras-transformed 3Y1 cells. Nucleotide sequence analysis showed that the NO3 protein is composed of 178 amino acid residues and does not have any sequence similarities with proteins in the data base. A putative zinc-finger domain is located in the central part of the sequence and a proline-rich domain is in the C-terminal region. NO3 mRNA was detected by Northern blot analysis in several tissues, including lung, kidney, intestine, and brain, but not in liver. Genomic Southern blot hybridization revealed that the NO3 gene exists as a single copy in the rat genome and that closely related, single-copy genes are also present in chicken and human.