Worm Breeder's Gazette 15(5): 40 (February 1, 1999)
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.
Department of Biochem. and Mol. Biophysics, HHMI, Columbia University College of Physicians and Surgeons, N.Y., N.Y. 10032
The synthetic Multivulva (synMuv) genes comprise two classes of genes that act in the determination of vulval precursor cell (VPC) fates (Ferguson and Horvitz, 1985, 1989). When a "Class A" mutation is combined with a "Class B" mutation, all VPCs adopt vulval fates and a Multivulva phenotype is seen. Single mutants and double mutants carrying two mutations of the same class have normal vulval development.
Under certain conditions, alleles of lin-13 display some of the genetic properties of a Class B SynMuv gene. Two alleles of lin-13, lin-13(n387) and lin-13(n388), were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985). Both mutations are heat sensitive and display maternal effects. At 25oC, lin-13 homozygous hermaphrodites segregating from a lin-13/+ mother are sterile and display a Multivulva phenotype, but at 15o they are fertile, non-Muv and grandchildless (i.e. their progeny are sterile). Ferguson and Horvitz (1989) provided evidence that lin-13 behaves like a Class B SynMuv by showing that lin-13(n387); lin-15A(n767) hermaphrodites are Muv at 15oC. We have obtained additional evidence, by finding that lin-38(n751); lin-13(n387) are also Muv at 15oC. Interestingly, we have found that lin-8(n111); lin-13(n387) hermaphrodites are not Muv at 15oC, which may explain why new alleles of lin-13 have not been isolated in screens requiring an interaction with lin-8.
Ferguson and Horvitz (1985) showed that lin-13(n387)/nDf16 is lethal, suggesting that lin-13(n387) is hypomorphic, and that the lin-13 null phenotype is zygotic lethality. To explore the issue of the lin-13 null phenotype, we used RNAi (Fire et al., 1998). We assayed the phenotype of the progeny of N2 hermaphrodites injected with dsRNA derived from lin-13 cDNA genomic clones. Injected mothers were incubated at 15oC or at 25oC. Progeny raised at 25oC were Muv and sterile, whereas progeny raised at 15oC were sterile but not Muv. Thus, the phenotypes caused by RNAi resemble those caused by the existing lin-13 alleles, which was a surprising result in view of the deficiency data.
We identified the molecular lesions associated with the two lin-13 existing alleles, and found that both are nonsense mutations. lin-13 encodes a large protein of 2,249 amino acids with 14 zinc-fingers of the C2-H2 class. lin-13(n387) is a TCA to TGA (S525stop) change that disturbs the first zinc-finger and would encode a predicted protein of 524 amino acids (Melendez and Greenwald, 1997). lin-13(n388) is a CGA to TGA (R707stop) change right after the second zinc finger and would encode a predicted protein of 706 amino acids. Thus, both nonsense mutations would result in greatly truncated LIN-13 protein products.
The RNAi and sequence analysis raise the distinct possibility that the null phenotype of lin-13 is the heat sensitive phenotype displayed by the two existing alleles, and that the behavior of lin-13 alleles in trans to nDf16 may be explained by the removal of another gene that interacts with lin-13. We are hoping to resolve the issue of the null phenotype of lin-13 by identifying an internal deletion.
Ferguson, E.L. and Horvitz, H.R. (1985) Genetics 110, 17-72.
Ferguson, E.L. and Horvitz, H.R. (1989) Genetics 123, 109-121.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Nature 391, 806-811.
Melendez, A. and Greenwald, I. (1997) International C. elegans Meeting abstracts, p. 366.