Worm Breeder's Gazette 12(5): 49 (February 1, 1993)

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More on lin-25

Simon Tuck, Iva Greenwald

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Dept. of Molecular Biology, Princeton University, Princeton, NJ 08544

lin 25 is required for proper vulval cell fate determination (Ferguson and Horvitz. 1985. Genetics 110 17-72). In animals carrying null mutations in lin-25 , P6 .padopts a vulval fate whereas all the other VPCs adopt the 3° fate (Ferguson et al., 1987. Nature 326. 259-267 and our own observations). Iin 25 mutations recessively suppress the multivulva phenotype of Iet-60 (gf)and lin-1 (lf)mutations but not the multivulva phenotype of lin-12 (d)allele. In a previous gazette article we described our genetic characterization of lin-25 (WBG 11 No. 5, 77); here we report our progress towards cloning lin-25 and some preliminary results from genetic mosaic analysis.

lin-25 cloning. We have identified a 10kb fragment that completely rescues the lin-25 mutant phenotypes (figure 1). When a plasmid containing 10kb subcloned from YL559 was injected at a concentration of 15mg/ml into animals homozygous for the temperature sensitive lin-25 mutation, n545 ,more than 80% of the F1 Rollers (n=100) (grown at the restrictive temperature) were rescued for the egg-laying defect. All the lines generated were also rescued. Deletion of 2kb from either end of the 10kb fragment abolished the rescuing ability.

lin-25 genetic mosaics. Temperature shift experiments indicate that lin-25 is required for the proper development of more than one component of the egg-laying system (Ferguson et al., 1987 and our own observations). We are scoring lin-25 mosaic animals, therefore, both for the vulval lineage defect and for the ability to lay eggs. To generate mosaics mutant for lin-25 in the AB lineage we constructed a strain of the genotype dpy-17 unc-36 ncl-1 ; unc-42 lin-25 (0); ctDp11 ( ctDp11 is-fusion between parts of chromosomes III and V; it complements an of these markers, Hunter and Wood. 1992. Nature 355. 551-555). We picked Unc NonDpy segregants, which were mosaics that had lost ctDp11 in AB or ABp. Half of them were Egl- (n=25). We have followed the vulval lineages in just two such mosaics so far (one AB mosaic and one ABp): both of these had wild-type vulval lineages and were Egl+. To generate P1 mosaics we constructed the strain sup-18 ncl-1 unc-36 ; unc-42 lin-25 (0); ctDp11 ; sup-10 ( n983 ).We picked wild-type segregants, which were mosaics that had lost c tDpl11 in P1 : sup-18 is a recessive suppressor of sup-10 ( n983 ),whose focus is in P1 .We have found two P1 mosaics so far; both of these were Egl-. There are several possible explanations for these results at the moment. Iin-25 might be required in two different tissues for proper egg-laying the VPCs (for proper fate determination) and a tissue derived from P1 (for some other, as yet unknown, aspect of egg-laying). The wild-type vulval lineages observed in the AB and ABp mosaics could be because of maternal rescue ( lin-25 homozygotes coming from a heterozygote are sometimes Egl+). A second possibility is that focus of lin-25 for proper vulval cell fate determination might be a tissue derived from both AB and P1 such as hyp7 .The focus for lin-15 for VPC fate determination is thought to be hyp7 (Herman and Hedgecock 1990. Nature 348. 169-171). We are currently examining the vulval lineages in more mosaic animals to distinguish these and other possibilities.

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Figure 1