TFIIIA GENE EXPRESSION IN OOCYTES AND SOMATIC CELLS

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The levels of the 5S RNA gene specific positive transcription factor, TFIIIA, appear to play a critical role in the developmental regulation of 5S RNA genes in Xenopus. The expression of the TFIIIA gene is known to be developmentally regulated at the level of mRNA accumulation. It is the objective of this proposal to identify the factors which control TFIIIA gene expression in oocytes and in somatic cells. This work is specifically directed at examining the molecular interactions involved in expression of-the TFIIIA gene in oocytes and somatic cells. In addition, differences between the expression of the TFIIIA gene in oocytes and somatic cells will be examined. Experiments to define cis-acting sequences of the TFIIIA gene and surrounding genomic DNA sequences, which affect the transcription of the gene, will be performed using standard gene fusion and transfection techniques. Deletion mutations of the TFIIIA gene and surrounding genomic DNA sequences will be prepared by in vitro mutagenesis protocols. The mutated sequences will then be assayed for the ability to direct specific transcription of the TFIIIA gene in oocytes and somatic cells. In this manner, it should be possible to define DNA sequences from the TFIIIA gene and surrounding genomic regions which act in a positive and/or negative fashion to control the expression of the TFIIIA gene in oocytes and somatic cells. Differences in the expression of the deletion mutants in oocytes and somatic cells will be examined carefully. These differences are likely to define regions of the TFIIIA gene and surrounding genomic sequences which are important in the tissue specificity of the TFIIIA gene. Finally, the interaction of cis-acting sequences which affect the expression of the TFIIIA gene with proteins (trans-acting factors) from oocytes and somatic cells will be examined using various DNA- protein binding assays. The presence or absence in oocytes or somatic cells or cell type specific modification of the DNA binding proteins may play a crucial role in the expression of the TFIIIA gene. Purification of the proteins which interact with cis-acting sequences which control the expression of the TFIIIA gene will be attempted. The purification of these proteins should allow the detailed examination of their role in the transcription of the TFIIIA gene in oocytes and somatic cells, including any modifications and tissue specificity.