Worm Breeder's Gazette 14(4): 63 (October 1, 1996)
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.
Dept. of Genetics, Harvard Medical School, and Dept. of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114
We are interested in the mechanism of action of the heterochronic gene, lin-14 in temporal control of cell fate. Basson and Horvitz (WBG, this issue) describe the isolation of a new heterochronic mutation, n2853, as a suppressor of the sterility of a lin-14(n179ts); egl-35(n694ts) mutant. n2853 animals display a retarded L/A switch defect (early adults lack alae) - opposite to the precocious phenotype of lin-14(n179) animals. We have shown that n2853 is epistatic to lin-14(n179) with respect to the L/A switch - at 25!C the double mutant does not display precocious adult alae and early adults lack alae. We reasoned that cloning of the n2853 locus could shed light on the function of lin-14. n2853 maps to the right arm of LGX, near unc-3 (Basson and Horvitz WBG, this issue). We showed that two small deficiencies that delete unc-3, mnDf5 and mnDf9, delete n2853. Since the n2853 mutant displays a temperature-sensitive (ts) lethal phenotype (Basson and Horvitz, WBG, this issue), we tested two let mutations that map close to unc-3, let-7(mn112) and let-9(mn107), for their ability to complement n2853. While let-9(mn107) complemented the lethality of n2853, let-7(mn112) failed to do so. This suggests that n2853 is a ts allele of let-7 and implicates let-7 in control of developmental timing. A closer examination of the canonical let-7(mn112) allele revealed that it displays a phenotype similar to let-7(n2853) - at all temperatures, let-7(mn112) animals explode at the vulva during the L4 to adult molt, lack adult alae, and males display a leptoderan tail (a spike protrudes from the fan). Our phenotypic analysis suggests a different age of death to that described previously for let-7(mn112) and establishes this mutation as heterochronic. We obtained a series of 8 cosmids from A. Coulson, that spanned from the immediate left to the immediate right of unc-3 (overlapping except for one gap). We injected let-7(n2853)/szT1 animals independently with two pools of 4 cosmids. Preliminary experiments indicate that the pool containing cosmids to the left of unc-3 (K01B4, C05G5, F33C8, F40E10), rescues the lethality of let-7(n2853) [2/2 lines], while the right-sided pool (F42D1, T04C10, C27C12, F31F6) does not [1/1 line]. We are currently injecting individual cosmids from the rescuing pool to identify a single rescuing cosmid. K01B4 alone does not rescue [2/2 lines]. The remaining 3 cosmids of the rescuing region have been sequenced by the Genome Sequencing Project and contain at least 15 genes as determined by Genefinder, seven of which have no homologs in the databases. One of the predicted genes (F40E10.2) found in the rescuing pool, is a transcription factor containing an HMG box with 95% identity to Sox-2 from humans, and 65% identity to SRY from humans. We are testing whether this Sox-2 homolog is let-7 for the following reasons: 1. HMG box factors act as cofactors in transcription; 2. let-7(n2853) has a similar phenotype to lin-29 mutants (see Basson and Horvitz, WBG, this issue); 3. lin-29 encodes a zinc-finger transcription factor, known to directly regulate the col-19 promoter; 4. the col-19 promoter contains four (1 perfect, 3 with one mismatch) consensus SOX binding sites (A/TAACAAA/T). We are sequencing let-7 alleles and have not identified any mutations in this gene so far. To identify additional genes that mutate to a similar phenotype as let-7(n2853), we have identified 14 more suppressors of the sterility of a lin-14(n179); egl-35(n694) mutant (30, 000 genomes screened). All of these mutations complement let-7(n2853), and may represent new genes. Additionally, we have obtained a suppressor of let-7(n2853) as a spontaneously arising mutation that increases the viability of let-7(n2853) animals, restores adult alae, and that alone causes a precocious alae defect. We are currently mapping all of these mutations and performing epistasis analysis with other heterochronic mutations to order them in the pathway.