A previously undocumented transition in embryonic gene activity has been identified in the amphibian, Xenopus. Genes transcribed by RNA polymerase III achieve a somatic pattern of expression by repressing selective templates during a gastrula-neurala transition (GNT). Our preliminary studies show that the GNT is complete by the onset of neurulation (60,000 cells) and is independent of transcription factor concentration. The proposed research program will be directed toward determining the mechanism responsible for this extensive and permanent repression of the previously active genes. Experiments will make use of an in RNA polymerase III chromatin transcription assay and will address the relationship between the changing embryonic cell cycle and regulation of gene activity. Embryonic chromatin will be isolated at regular intervals to determine the exact timing of the GNT. The temporal order of gene-specific replication will be.examined with respect to the decline in transcriptional activity during the transition period. The cause and effect relationship between replication timing and gene expression will be addressed by increasing the number of transcriptionally active oocyte 5S RNA genes via microinjection of the MRNA for the 5S RNA gene-specific transcription factor TFIIIA. We can then determine whether replication timing of these normally inactive genes is altered by placing these sequences in an active conformation. The potential role of histone HI in controlling gene expression during the GNT will be tested by microinjection of synthetic HI MRNA to effectively increase the concentration of the HI protein in vivo. Finally, an in vitro assay system which reproduces the entire progression of regulated gene activity will be developed. This will permit a subtractive analysis by immunodepletion along with other reductionist approaches.
| Pittman, R H; Andrews, M T; Setzer, D R (1999) A feedback loop coupling 5 S rRNA synthesis to accumulation of a ribosomal protein. J Biol Chem 274:33198-201 |
| Frederick, D L; Andrews, M T (1994) Cell cycle remodeling requires cell-cell interactions in developing Xenopus embryos. J Exp Zool 270:410-6 |
| Rollins, M B; Del Rio, S; Galey, A L et al. (1993) Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos. Mol Cell Biol 13:4776-83 |
| Leverette, R D; Andrews, M T; Maxwell, E S (1992) Mouse U14 snRNA is a processed intron of the cognate hsc70 heat shock pre-messenger RNA. Cell 71:1215-21 |
| Rollins, M B; Andrews, M T (1991) Morphogenesis and regulated gene activity are independent of DNA replication in Xenopus embryos. Development 112:559-69 |
| Andrews, M T; Loo, S; Wilson, L R (1991) Coordinate inactivation of class III genes during the Gastrula-Neurula Transition in Xenopus. Dev Biol 146:250-4 |
