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.

No 5-Methyl-Cytosine in Worm DNA

V.J. Simpson, T. Johnson

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.