This application entails the analysis of a C. elegans gene, pag-3, which apparently encodes a transcription factor suppressing touch receptor (ALM) cell fate. In preliminary experiments carried out in the investigator's lab, the pag-3 mutation was identified by its ability to ectopically express touch receptor specific genes (e.g., mec-7, mec-4) in other neurons (e.g., the BDU interneurons). In the past year the investigator has cloned the gene and showed that it encodes a transcription factor of the C2H2 zinc-finger family. Beside the misexpression of genes, pag-3 mutants have a behavioral defect (""""""""reverse kinker Unc phenotype"""""""") and variable abnormalities in the axonal projection of ALMs and BDUs. All proposed experiments revolve around the further characterization of this gene. A. The expression of pag-3 will be studied by a promoter construct driving lacZ or GFP (green fluorescent protein) expression, as well as immunohistochemically by an antibody produced against a fusion protein. B. Genetic mosaic analyses will be carried out to address the question in which cells pag-3 is required. Strains were constructed in which a duplication bearing a wild-type copy of pag-3 is linked closely to marker mutations (osm-1, preventing FITC uptake by various cells; sup-10, suppressing the """"""""rubberband phenotype) and is expressed in the pag-3 mutant background. Mosaics will be generated in which the duplication, which rescues the pag-3 phenotype, is lost in specific lineages. Specific questions which can be addressed are whether muscle cells or neurons need to express pag-3 (to avoid the reverse kinker phenotype), and which cells need to express pag-3 to avoid misexpression of ALM specific markers. C. The function of pag-3 will be addressed by: 1) trying to replace pag-3 by its putative mammalian homolog, Gfi-1.; 2) carrying out in vitro studies (DNAse I footprinting) of PAG-3 protein to the control regions of ALM specific genes which are misexpressed in the pag-3 mutant background. These experiments are intended to establish whether the effect of PAG-3 on these genes is a direct one; 3) expressing pag-3 ectopically. Using an unc-86 promoter, pag-3 will be specifically expressed in the ALM precursors to ask whether this can block ALM fate; in a second experiment, pag-3 will be generally expressed under heatshock control; 4) Expression of pag-3 in the - background of mutations in all (11) genes known to be required for ALM development will be analyzed; stocks carrying double mutations in these genes and pag-3 will be analyzed.
| Toms, N; Cooper, J; Patchen, B et al. (2001) High copy arrays containing a sequence upstream of mec-3 alter cell migration and axonal morphology in C. elegans. BMC Dev Biol 1:2 |
| Aamodt, E; Shen, L; Marra, M et al. (2000) Conservation of sequence and function of the pag-3 genes from C. elegans and C. briggsae. Gene 243:67-74 |
| Jankowska-Anyszka, M; Lamphear, B J; Aamodt, E J et al. (1998) Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures. J Biol Chem 273:10538-42 |
