Worm Breeder's Gazette 16(1): 35 (October 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.
|1||Department of Biology, Santa Clara University, Santa Clara, California 95053|
|2||Present address: Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven Connecticut 06536|
The lin-31 gene encodes a winged-helix transcription factor involved in specifying vulval cell fates (Miller et al., 1993, Genes and Development 7:933). The LIN-31 protein contains a DNA-binding domain, an acidic region, a serine-rich region, and a proline-rich region. To elucidate possible functions of these domains, the 4 kb genomic domain spanning the lin-31 coding region was sequenced in 16 lin-31 mutant alleles. This sequencing revealed 6 nonsense mutations, 3 deletions, 4 transposon insertions, 1 frameshift mutation, and 2 missense mutations. All six nonsense mutations were found in the DNA binding domain. No late nonsense mutations were observed, indicating that a LIN-31 protein truncated late in the protein may not result in a phenotype detectable by standard Muv and Vul screens. The two missense mutations are located at conserved residues in the DNA-binding domain. It is important to determine if the amino acid substitutions result in a destabilized protein or if the encoded mutant proteins are defective because they are no longer able to bind their DNA target. We have ruled out the first possibility by using antibody-binding experiments to show that the LIN-31 protein is present in the proper cells at the proper stage of development. Because all (or almost all) existing lin-31 alleles display a similar phenotype, these studies show that (1) the null phenotype of lin-31 is the phenotype displayed by most, if not all, of the existing alleles, (2) direct screens for multivulva and vulvaless mutants will probably only yield null (or strong) alleles of lin-31, and (3) the DNA-binding domain plays a critical role in LIN-31 function.
To confirm that the LIN-31 proteins encoded by these two mutant alleles are defective in DNA binding, we are performing in vivo extrachromosomal array binding experiments that utilize lac operator/repressor recognition (Carmi et al., 1998, Nature 396:168; Gonzalez-Serrichio and Sternberg, manuscript in preparation; Straight et al., 1996, Current Biology 6:1599). We are constructing transgenic strains whose extrachromosomal arrays carry multiple copies of the rat transthyretin (TTR) promoter (a known HNF-3 winged-helix transcription factor binding target), lac operator (lacO) and transgenes encoding GFP-tagged lac repressor driven by a heat-shock promoter (hs-LacI::GFP). Binding of tagged lac repressor to lacO sequences allows us to tag the TTR promoter in vivo. Failure of marked TTR and antibody-stained mutant LIN-31 to co-localize to the array would indicate that these LIN-31 mutant proteins fail to bind their DNA target. In a preliminary experiment, a control strain (courtesy of Ilil Carmi and Barbara Meyer) containing lacO, hs-lacI::gfp, and rol-6 was analyzed to determine if extrachromosomal arrays could be observed as discrete entities in post-embryonic cells. After heat shock of this strain, extrachromosomal arrays were observed at the correct stage for scoring LIN-31 binding (L2 and L3 larval stages). By fluorescence microscopy, one to three copies of the array were clearly visible in the nucleus of each cell, indicating that this experiment will be possible in post-embryonic animals.
In addition, we are expanding our structure/function study of LIN-31 by conducting site-directed mutagenesis experiments to elucidate how the acidic, serine-rich, proline-rich, and other regions may contribute to the function of the LIN-31 protein.