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.