Two kinds of 5S RNA multigene families exist in the genome of Xenopus. The oocyte-type 5S RNA genes function only in the oocyte and are repressed in somatic cells. The somatic 5S RNA genes are transcribed actively in both cocytes and somatic cells. The expression of these genes can be assayed by the synthesis of 5S RNA in vitro, in vivo, or with isolated chromatin. The goal of these experiments is to reproduce in vitro with purified components the same differential control of gene activity that is seen in vivo. In somatic cells of Xenopus, active 5S RNA genes are associated with transcription factors to form stable transcription complexes. Repressed genes are not associated with these factors but rather complexed with nucleosomes that keep transcription factors from forming complexes with the gene. Both the active and repressed state of 5S RNA genes are stable. One purpose of this project is to identify the molecules associated with active and repressed complexes and characterize the details of how they interact with each other and with DNA signals in and around the 5S RNA gene. The gene for the 5S-specific positive transcription factor (TFIIIA) will be cloned, expressed in E. coli, and mutants prepared in order to map further the functional amino acids of the protein that bind to critical regions of the internal control region of the 5S RNA gene and to other components of the transcription complex. A transient developmental expression assay is sought that will reproduce the control of the two kinds of 5S RNA genes that occurs during early embryogenesis. From this assay, the exact DNA sequences in the 5S RNA gene involved in the developmental control, as well as the molecules in embryos that are responsible for reading the DNA signals, will be elucidated. In addition, this assay will be designed to determine how discrimination between two genes is established and then perpetuated in early embryogenesis. It is suggested that these stable transcription complexes might play a role in the phenomenon of determination or """"""""commitment"""""""" of genes in development. Some experimental schemes will test this proposal.
