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.

The heterochronic mutation n2853 is an allele of let-7: complementation, molecular analysis, and rescue.

Frank Slack, Brenda Reinhart, Gary Ruvkun

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.