Worm Breeder's Gazette 12(5): 16 (February 1, 1993)

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.

ncl-1 to the Rescue

David Waring

Fred Hutchinson Cancer Research Center, 1124 Columbia St, Seattle
WA 98104

Mutations in the gene ncl-1 cause many cells in the worm to have unusually large nucleoli. This phenotype and the fact that ncl-1 acts cell autonomously has allowed the ncl-1 gene to be used as a marker for mosaic analysis by various groups.

The traditional method for obtaining genetic mosaics, has been to make a strain with mutant alleles of the gene(s) of interest on the chromosomes, and wild type alleles on a free duplication (which is lost during mitosis at a relatively low frequency). Using appropriate marker mutations which are also covered by the same duplication one can identify animals which have lost the free duplication in a subset of cells. When ncl-1 is used as a marker, it is possible to look at an individual cell, under Nomarski optics, and determine its genotype (whether or not it contains the duplication) by examination of the nucleolus. Thus using ncl-1 as a marker can allow single-cell resolution in mosaic analysis. This technique requires that the gene of interest be linked genetically to the ncl-1 gene, and any other markers. This is a severe limitation, as most genes are not on chromosome three.

At least two groups have gotten around this problem by artificially linking the genes that they wanted to study to ncl-1 .Hunter and Wood (Nature 1992) did this by linking two duplications together using x-rays thus creating one large free duplication. This of course makes all genes which are covered by this duplication pseudo-linked. Leung-Hagensteijn et al. (Cell 71;289) generated a strain in which the cloned unc-5 gene had been integrated onto the duplication sDp3 which carries ncl-1 .

In order that ncl-1 might be even more readily used as a marker for mosaic analysis of other genes I have begun an attempt to clone the. ncl-1 gene. Mapping data from Chisolm, and from Kenyon, placed ncl-1 between the genes lin-39 and mab-5 By first injecting pools and then individual cosmids I have rescued ncl-1 ( e1942 )with the cosmid C33C3 (from England).

It should now be possible to artificially link ncl-1 to any cloned gene. Extrachromosomal arrays of injected DNA apparently behave like free duplications. By generating an extrachromosomal array containing the gene of interest, ncl-1 and any other cloned marker genes or gene fusions, one should be able to use this array in the same manner that free duplications have been used. It remains to be seen if such an array is lost at an appropriate rate, and if the mosaism that has been observed with extrachromosomal arrays is due solely to mitotic loss or rather to mosaic expression. It should also be possible to integrate the cloned ncl-1 gene onto a free duplication which covers a gene of interest, and in this way link the two genes, in much the same way that Leung-Hagensteijn et al. linked ncl-1 and unc-5 .In this case it would not be necessary to clone the gene of interest if it is covered by a free duplication.