We have visualized extrachromosomal arrays by targeting the green fluorescent protein (GFP) to a specific DNA sequence (lac operator) incorporated into Caenorhabditis elegans' transgenes. This system can be used to determine polyploidy and to investigate chromosome segregation. This technique also allows rapid, accurate determination of spontaneous loss of an array, thereby allowing high-resolution mosaic analysis. We carried out genetic mosaic analysis on lin-3 (epidermal growth factor) using the GFP-Lacl + lacO method. This methodology confirmed lin-3's site of action for vulval induction is at the anchor cell. This result also proved this technique works.
We used both the GFP-Lacl + lacO256 system as well as the ncl-1 gene as genetic mosaic markers to determine the site of action of lin-15A and lin-15B. Both markers indicate that lin-15A gene function is required within the vulval precursor cells (VPCs) to prevent an excessive number of VPCs from generating vulval progeny. The mosaic expression pattern for lin-15B is broad therefore, proven difficult to pinpoint a site of action.
The products of the lin-15 gene were first defined genetically as negative regulators of the vulval induction pathway. It encodes two novel hydrophilic proteins, LIN-15A and LIN-15B. According to antibody stainings and GFP expression patterns, both proteins are nuclear and present in almost all the cells. lin-15 is part of the synthetic multivulva (synMuv) set of genes which are comprised of two classes, A and B. Mutation of both an A and a B gene is required to obtain a multivulva (Muv) phenotype. Further characterization of the lin-15 locus reveals an effect on fertility.</p
