Worm Breeder's Gazette 8(3): 16
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.
Chromatographic analysis of DNA from several strains of C. no detectable 5-methyl-cytosine at any age from 0-16 days. Age-synchronous cultures were started by hypochlorite treatment of large asynchronous populations harvested from chicken egg plates. Isolated nematode eggs were hatched and grown on NGM plates for three days with daily feeding of E. coli. At three days they were transferred to aerated, mass culture bottles, they were maintained in S. Basal with a bacterial concentration of 3 x 10+E9 cell/ml. Reproducing populations were kept age-synchronous by daily filtration on Nytex filters which separates adults from larvae. Worms were washed free of bacteria and debris at specified times by spinning through 50% sucrose and collecting worm band on top. DNA was isolated by the standard SDS/proteinase K procedure. After ethanol precipitation the DNA was further purified by equilibrium centrifugation on cesium chloride. Purified DNA was hydrolyzed to free bases in 88% Formic acid at 120 C for one hour. 5-methyl- cytosine is stable under these extraction conditions. The formic acid was evaporated off under a stream of N2. The acid-digested DNA was analyzed by high pressure liquid chromatography (HPLC) using an Altex ODS-IPC18 column and monitoring absorption at 254 and 280 nm. Samples were compared to authentic standards. Limits of detection of 5-methyl-cytosine were approximately 0.01 mole%. We detected no 5-methyl cytosine peak in DNA isolated from L1 or dauer stage worms. In older cultures (10-16 days) we observed a peak which appeared within 20 secs of an authentic 5-methyl-cytosine standard on a 10 min HPLC chromatogram. These 10, 15 and 16 days old DNA samples were further characterized using Waters-varian HPLC system which allows a UV scan of isolated peaks. Scans (190-340 nm) were performed on the peak in question and did not match scans of authentic 5-methyl-cytosine. Therefore 5-methyl-cytosine has not been detected in worm DNA at any age to within our detection limits of 0.01 mole%. Isoschisomer analysis of old worm DNA using the enzymes HpaII ( doesn't cut C(m)CGG) and MspI (cuts C(m)CGG) and probed with a variety of probes including Tc1 (pCe2002) failed to detect 5-methyl-cytosine. The earlier report of the accumulation of 5-methyl-C in concentrations up to 14 mole% in the DNA of aging C. e appears to be in error, perhaps due to the use of DNA not purified by CsCl and containing a contaminant which eluted near authentic 5-methyl-C.