The proteins which regulate transcription of the ribosomal RNA genes of Xenopus laevis will be investigated using several approaches. DNase I protection, or footprinting, of the promoter shows three protein binding sites which correspond closely with sequences which are necessary for transcription initiation. This assay will be used to isolate the sequence-specific DNA binding protein(s) conferring protection on the promoter of this gene. The relationship of the binding sites to each other and the mechanism of transcription complex formation will be studied by further binding experiments. Monoclonal antibodies will be made to track the protein(s) through the developmental changes which affect the activity of this gene and to map the functional domains of the protein. The role of the sequence-specific DNA binding protein(s) in transcription will be studied by using an oocyte injection assay. In order to relate the DNase I protection patterns observed in vitro to the in vivo structure of the genes, potential factor binding sites will be mapped at high resolution by digesting the endogenous gene with DNase I and analyzing the products using an indirect labeling scheme, which will yield an in vivo footprint. The DNA-binding proteins associated with the amplified nucleoli in the oocyte during the transition from inactive to active and the resulting chromatin conformation will be examined and correlated with gene activity.
| Dunaway, M; Trason, A (1990) Reprogramming of the transcriptional machinery in Xenopus oocytes by injection of mouse poly(A)+ RNA. Mol Cell Biol 10:6055-8 |
