Worm Breeder's Gazette 11(5): 68
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.
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 duplications. 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.