Worm Breeder's Gazette 9(2): 67

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.

Sperm DNA Strands Do Not Co-Segregate in the Next Generation

K. Ito and J. McGhee

By feeding male worms on bromodeoxyuridine (BrdU) labelled E.  coli 
and using a monoclonal antibody to BrdU, we have been able to identify 
paternal DNA strands within the next generation of developing embryos. 
The following is our current protocol, although it is still being 
A thymidine requiring E.  coli strain was grown to saturation in M9 
salts supplemented with 0.4 % glucose, 1mM MgSO4, 1.25  g/ml vitamin 
B1, 9  M BrdU and 1  M thymidine.  The culture was concentrated and 
ampicillin was added so as to prevent contamination of plates with 
unlabelled bacteria that the worms seem to find tastier.  A mixed 
population of males and hermaphrodites (N2) at dauer larva stage were 
transferred onto M9-agarose plate with this BrdU-labelled E.coli.  
After two days at 20 C, male worms were transferred onto a new M9-
plate, and were continued being fed on BrdU-bacteria for an additional 
two days.  The incorporation of the label can be seen marching down 
the gonad over a period of a few days.  The labelled males were mated 
to spermless hermaphrodites, fem-2, (raised at 25 C) on a NGM-plate 
with non-labelled E.  coli (OP50).  The hermaphrodites also carried a 
spontaneous dumpy mutation that turned out to be convenient for 
separating unlabelled hermaphrodites from any contaminating labelled 
hermaphrodites inadvertantly brought in with the males.  After about 
five hours of mating, dumpy hermaphrodites were picked up and cross-
progeny were obtained by cutting the worms in half followed by gentle 
hypochlorite treatment (0.4-0.6 % NaOCl in egg-salts, 3 min.).  The 
embryos, from which egg-shells were removed by chitinase-chymotrypsin 
digestion, were transferred onto a subbed slide and gentle pressure 
was applied on the cover-slip to spread the cells to avoid cell-cell 
overlap as well as to permeabilize the embryos.  To detect the 
labelled DNAs, we used monoclonal anti-BrdU antibody conjugated with 
fluorescein (Becton-Dickinson).  Prior to antibody binding, DNA was 
denatured in 6N HCl for 3 minutes, followed by neutralization in a 
large excess of 0.4M borate buffer, pH 8.5 for ten minutes.  After 
antibody binding (30  l of a 1:10 dilution per slide), the DNA was 
stained with DAPI.  Distinct green spots appear within the embryos 
under epifluorescence.  Straightforward controls seem to work; green 
spots are not seen in unlabelled worms; antibody is competed off with 
BrdU; green spots go away with DNase I.
In embryos with total cell number of 100 or more, the number of 
green spots seems to converge on 10 +/- 2.  Our initial conclusions 
are that: (1) sister chromatid exchange does not appear to occur at 
high frequency, and (2) sperm DNA strands do not co-segregate, since 
this would predict only two labelled cells in embryos of all ages.  A 
few preliminary experiments with larval worms suggest that strand 
segregation could be random, i.e.  green spots appeared at different 
positions in different larvae.  We should be able to test far more 
rigorously whether DNA strand segregation is truly random.  Further 
experiments should be possible by variation on the above technique; 
for example, following maternal DNA strands, following both paternal 
and maternal strands in the same embryo, following strand segregation 
in post-embryonic cell divisions and so on.