Thmor - suppressive Activity of N 03 Gene Product in v - src - transformed Rat 3 Y 1 Fibroblasts '

in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Cells were grown at 37°Cin a humidified 5% CO2 atmosphere. †̃fransfection. Thefull-lengthN03cDNAwas insertedintotheEcoRlre striction site of pMEXneo expression vector (13) in the forward orientation. The expression vector containing N03 cDNA or the expression vector alone was introduced independently into SR-3Y1 cells by using the lipofectin method (14). Cells were maintained in the presence of400 @Wml 0418 (Sigma Chemical Co., St. Louis, MO). Two weeks later, clones showing G418 resis tance were isolated and subsequently analyzed. Northern Analysis. Total RNA (10 @g) was electrophoresed in 1% agarose/formaldehyde gels, transferred to nylon membrane filters, and immo bilized by UV cross-linking. Hybridization probes were made from gel-puri fled DNA fragments that were labeled by random priming with the Klenow fragment of DNA polymerase and [a-32P]dCFP (14). After hybridization at 42°Cfor 20 h in 6X (SSC) containing 5X Denhardt's solution, 0.1% SDS, 50% formamide, and 100 @gof heat-denatured salmon sperm DNA/mI, the filters were washed sequentially in 2X SSC/0.1% SDS at room temperature for 30 mm and in 0.1X SSC/0.1% SDS at 50°Cfor 30 mm. Autoradiography was for 2 days at —70°C with an intensifying screen. Southern Hybridization. High molecular weight genomic DNA was di gested completely with appropriate restriction enzymes and size-fractionated in 0.8% agarose gels. The DNA was transferred to nylon membrane filters, which then were prehybridized for 6 h at 50°Cin 6X SSC, 5X Denhardt's solution, 0.1% SDS, 50% formamide, and 100 @&g of heat-denatured salmon sperm DNA/mI. Random hexamer-primed DNAS were radiolabeled and hybridized with membrane-bound DNA for 20 h.