The goal of this proposal is to develop a general strategy for discovering new components in transcriptional regulatory networks, using Dictyostelium discoideum as an experimental system. The plan is to use fluorescence activated cell sorting (FACS) to enrich for mutant amoebae which show altered expression of a reporter gene, green fluorescent protein (GFP) under the regulation of two developmentally responsive promoters controlling the cAMP receptor gene 1 (CAR1) and the prespore specific antigen (PsA). The investigator will mutagenize amoebae first by chemical means, and later by restriction enzyme mediated integration (REMI) of a plasmid, and then enrich for cells with precocious expression of developmentally regulated genes. He plans to recover mutated trans-acting genes tagged with the plasmid and from them, their wild type counterparts. The expectation is that such genes will be involved in the transcriptional apparatus or in signal transduction. In preliminary experiments the investigator has used flow cytometric analysis to identify cells expressing actin 15-lacZ fusion genes using a fluorescin C2- di-beta-D-galactoside (FDG) substrate. Although the fluorescence signal was above the background of autofluorescence and the endogenous beta-galactosidase activity, the sensitivity is relatively low. Instead with an actin 15-GFP plasmid, cells expressing the reporter gene could be detected with 10-fold greater sensitivity than with the actin 15-lacZ fusion gene. The investigator proposes to use the CAR1 promoter or the DR53/D19 promoter for a prespore glycoprotein PsA fused to GFP. He prefers to use the CAR1 promoter because it is activated early in development in an autocrine mechanism and dependent on pulsatile extracellular cAMP signaling. It is repressed by high extracellular cAMP and regulated by folate. Although the promoter is complex, the part of it that he plans to use controls the early expression and not the late expression of CAR1. Preliminary results show that the vegetative cells expressing the CAR1 regulated GFP can be distinguished from the background of cells which are undergoing autoflluorescence. Later experiments will use the PsA-GFP with cells that have started development. This gene is normally expressed after 8 hrs in development. With PsA-GFP, he expects that he initiates the developmental program prior to sorting, he will more likely find mutants in which mutated trans-acting factors might result in precocious expression of the gene than he used vegetative cells. To improve the sensitivity of the method he plans to incorporate modifications of the GFP amino acid sequence (Ser65 to Thr) reported by Tsien, which enhance the absorption/excitation at 490 and increase the rate of conversion to the fluorescent species in E. coli. He proposes to mutagenize cells chemically with NTG first or with REMI later. Mutagenesis using REMI tags the genes for easy isolation and characterization, but the yield of mutant cells is low relative to NTG mutagenesis.
| Nadin, B M; Mah, C S; Scharff, J R et al. (2000) The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein. Dev Biol 217:173-8 |
