Worm Breeder's Gazette 12(1): 32 (September 1, 1991)

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.

Mosaic analysis of unc-5 using an unc-5 (+)cosmid array fused to sDp3

Joe Culotti[1], Ed Hedgecock[2], Andrew Spence[3]

[1]Mt. Sinai Hospital, Research Institute, Toronto M5 G1X5
[2]Biology, Johns Hopkins U., Baltimore, MD 21218
[3]Molecular & Medical Genetics, U. of Toronto

We have been able to construct a strain suitable for mosaic analysis of unc-5 by fusing the unc-5 cosmid array eEx15 / unc-5 (+)/to sDp3 / dpy-1 (+) ncl-1 (+)/using the technique described by Herman and Kari (WBG11#5, p.68). Briefly, we first constructed strain NW705 which carried eEx15 and sDp3 as independently segregating free Dps in a background that was chromosomally mutant for dpy-1 ncl-1 ,and unc-5 .This strain segregated wild type, Unc nonDpy, Dpy nonUnc, and Dpy Unc progeny. To attempt to fuse eEx15 and sDp3 we treated NW705 adults with 3600 rads of gamma-rays from a [137Co] source. 894 F1 progeny were cloned of which 344 produced broods of greater than 50. Among these we found 9 independently derived strains that behaved genetically as though the cosmid array eEx15 had been fused to sDp3 ,i.e., they segregated wild type and Dpy Unc self-fertilization progeny, but no Unc nonDpy or Dpy nonUnc progeny. (Some did initially segregate a few Dpy nonUnc progeny, but siblings that only segregated wild types and Dpy Uncs could easily be found, suggesting that extra unfused copies of the cosmid array can hang around).

To assess the frequency of mitotic loss of the fused Dp in these 9 strains, we looked for 'gonad mosaic' animals that moved normally, but had 1 or more misreflexed gonad arms (Hedgecock et al., Neuron 4, 61-85,1990). For 6 strains we failed to see any such mosaic animals and for 2 strains the frequency of such mosaic animals was judged to be too high (approximately 1% or more) to be useful for mosaic analysis. For 1 strain, however, the frequency of mosaic animals was less than 1 in 500, so we chose this strain, NW707 for mosaic studies and named the fused Dp evEx8 .

This strain has proven to be useful for mosaic analysis of unc-5 .By scoring nucleolar size in cells derived from a number of embryonic blast cells, we have found a number of mosaic animals which are all consistent with cell-autonomous action, that is, unc-5 acts in cells that normally exhibit dorsal migrations on the epidermis (motorneurons DA, DB, DD, AS and VD; mesodermal distal tip cells; ectodermal excretory cell). This result is consistent with the hypothesis that the unc-5 protein is an adhesive receptor present on migrating mesodermal cells and neuronal growth cones (CSH Abstracts, 275-276, 1989).