In order to investigate the role of LIN-3 in cell fate specification, we conducted a F2 screen using a reduction of function
lin-3(
n378) strain which contains an integrated transgene that carries a mutation that presumably eliminates
lin-3B splicing and should, therefore, only produce
lin-3A. The line carrying the integrated transgene is fertile and 100% multivulva. Animals that lack
lin-3A are sterile (Jing Liu & PWS, unpubl.). To potentiate rescuing any lines that are sterile because they lack
lin-3A,
lfe-1 was incorporated into the strain background.
lfe-1(
sy290) is a gain of function allele that partially rescues the sterility caused by
let-23 loss of function animals. The strain is fertile and 100% multivulva. From these screens, we looked for animals which were non-Muv and isolated two mutations. One of these mutations still displayed some induction, but displayed neither invagination nor morphogenesis. Since these animals were 100% vulvaless, we artifically created a hole connecting the outside to the uterus using a typical injection scope and needle. After obtaining cross progeny and outcrossing this mutant, it displayed characteristic heterochronic defects; extra molts, late divisions, and no adult cuticle. Having preliminarily mapped this mutation to linkage group II, we tested the hypothesis that it was a
lin-4 allele by performing single worm PCR on the homozygous animals using primers to
lin-4. Indeed, there was a mutation in the
lin-4 gene, C516T. Though there are only two other alleles of
lin-4 ,this is the second time that this mutation has been isolated (the other allele was isolated in Victor Ambros lab and is known as
ma161). Lin-4 is a heterochronic retarded mutant which is critical to the controlled coordination of cell division in postembryonic divisions. What is most interesting about
lin-4 is that it encodes a 22 nt. RNA form to block translation of other proteins such as
lin-14 and
lin-28. The fact that this allele has been isolated twice is indicative of the importance of this site in the function of the
lin-4 RNA. It has been hypothesized (Ha, I. and colleagues) that this C forms a bulge which is recognized by another, as of yet unknown, factor. When this C is replaced with a T, this bulge does not form, and does not inhibit the translation of
lin-14 and
lin-28.