Worm Breeder's Gazette 9(1): 14
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The existance of cellular oncogenes in C. elegans was investigated. DNA was isolated by the method of Emmons (Gazette, Vol. 3, # 2, p. 34) from the eggs of C. elegans. About 0.5 g of eggs isolated by treatment with hypochlorite from about 5x10+E4 adults were suspended in 2.5 ml of a solution containing 0.1 M NaCl, 0.1 M EDTA, 0.05 M Tris- HCl (pH 8.0) and were ground to a powder in a mortar on dry-ice. Proteinase K (Sigma) was added to 200 r/ml and the mixture was incubated overnight at 37 C. 300 l of the mixture was suspended in 3. 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 3.7 g CsCl were added. After centrifugation at 32,000 rpm for 48 hours, the purified DNA was obtained. DNA samples were digested with the restriction enzyme EcoRI and subjected to agarose gel electrophoresis (8%, 20 V, 20 hours) and filter-blot transfer to Zeta-Probe Blotting Membrane (Bio Rad). Filter-blotted DNA was prehybridized(#1) and hybridized(#2) with a nick-translated [alpha-P32]dCTP-labelled DNA probe (K-ras and V-myc genes) under two sets of conditions, nonstringent(#3) and stringent(#4) hybridization. Hybridization was revealed by autoradiography for two days at -70 C (XAR-5 film, Kodak). One K-ras and three or four V-myc oncogenes were identified in C. elegans.1) Incubation was performed in a solution containing 5x SSC, 0.05 M sodium phosphate buffer (pH 6.5), 10x Denhardt's, 0.5 mg/ml salmon sperm DNA, 50% folmamide, 5% Dextran sulfate (M.W. 5,000) for ten minutes at 85 C and overnight at 42 C. 2) Incubation was done in a solution 5x SSC, 0.05 M sodium phosphate buffer (pH 6.5), 2x Denhardt's, 0.2 mg/ml salmon sperm, 50% folmamide, 10% Dextran sulfate for ten minutes at 85 C. 3) Incubation was done in 2x SSC, 0.1% SDS for 60 minutes at 65 C and shaken overnight at room temperature. 4) After the nonstringent hybridization, incubation was done in 0. 1x SSC, 0.5% SDS for 120 minutes at 68 C. After the solution was changed, incubation was continued for a further 60 minutes at 68 C.