Worm Breeder's Gazette 11(5): 68

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Fusing Nonhomologous Free Duplications

R.K. Herman and C.K. Kari

Ed Hedgecock has shown that ncl-1 III is a lovely cell marker for 
mosaic analysis.  A ncl-1 mutation results in large nucleoli that can 
be scored in nearly every cell of living animals; the mutation behaves 
cell autonomously in ncl-1 mosaics; and there is no detectable 
perdurance of wild-type gene product following loss of a ncl-(+)-
containing free duplication during embryogenesis.  Several people have 
made use of ncl-1 as a cell marker in their mosaic studies.  But these 
studies have all been limited to genes that are linked to ncl-1, in 
order that both the wild-type gene of interest and ncl-1(+) are 
carried by the same free duplication.  Clearly, it would be nice to be 
able to put ncl-1(+) on free duplications covering other regions of 
the genome.  One approach would be to persuade a cloned wild-type ncl-
1 gene to attach to free duplications.  Some work we did with Andy 
Fire (WBG 11(3):14) on using gamma rays to fuse an extrachromosomal 
array to a free duplication could be useful in this connection.  An 
alternative approach that we've begun to explore is to fuse a ncl-1(+)-
containing free duplication to a free duplication that carries the 
gene one wants to study in mosaics.  The idea for this approach came 
from the work we did with Andy.  We have simply used gamma rays to 
promote the fusion of sDp3[ncl-1(+)] with nonhomologous free 
We've done two sets of experiments.  In the first set, we made dpy-1 
sDp3(III;f)[dpy-1(+) ; 
mnDp30(X;f)[unc-6(+)] hermaphrodites, which, as 
expected, showed independent meiotic segregation of the two free 
duplications.  We treated these animals with 3,800 r of gamma rays, 
picked individual wild-type F1 progeny, and screened for broods 
containing only wildtype and Dpy   One (out 
of 538 broods screened) was found and outcrossed.  In the second set, 
we treated dpy-1 sDp3; mnDp14(X;
f)[unc-3(+)] hermaphrodites in analogous fashion and found five 
independent fusions (out of 414 broods screened; the higher success 
rate in this case is probably due, at least in part, to the much 
greater relative meiotic stability of mnDp14 compared with mnDp30).  
All six of our fused duplications carry ncl-1(+), but all are 
considerably more stable mitotically than either sDp3 or mnDp14 (each 
of which shows a convenient frequency of loss of about 1/400 per cell 
division at 20 C).  We are guessing that the enhanced mitotic 
stability is due primarily to increased duplication size (we looked at 
two of the sDp3mnDp14 fused duplications cytologically and they did 
seem large).  Perhaps the duplications are fused end-to-end; 
translocated duplications seem to show a strong tendency to attach to 
chromosome ends.  We are now beginning to prune sDp3 (retaining ncl-1(
+) and a closely-linked marker) and to prune one of the fused 
duplications to see if we can generate duplications that would be 
better for producing mosaic animals.