Objective. The long term goal of the proposed research is to understand the molecular mechanisms by which Class III genes are differently regulated. During the previous grant period, we demonstrated that sequence differences preceeding the somatic and oocyte 5S promoters effect the relative activities of these genes in vitro and in vivo. This modulatory effect is only observed under conditions whereby the somatic 5S gene has a large (100-200 fold) transcriptional advantage over oocyte 5S. We have shown that these sequence differences increase the affinity for a common transcription factor and thereby aid in stable complex formation. We have further demonstrated that of the common factors IIIA, B, and C, the affinity of TFIIIA is not affected by these sequence differences. This finding is significant since it suggests that the differential expression of these genes is mediated not only by the 5S specific factor TFIIIA, but also through preferential association of IIIB with the somatic 5S gene. We now wish to determine which factor(s) preferentially interacts with the somatic 5S gene as a result of these sequence differences. We also wish to determine why these sequence differences effect gene activity only in whole oocyte S150 extracts in vitro (not in nuclear extracts) and at certain developmental stages in vivo. Is this indicative of stage specific forms of the polymerase III factors? In a related finding, we have shown that two other Class III genes, tDNAmet and OAX, are also differentially expressed in vitro in whole oocyte and cocyte nuclear extracts. Most interestingly, these DNAs compete with 5S DNA for factors in the nuclear extracts but not in the whole oocyte extract. We wish to determine if this finding reflects multiple gene specific forms of the polymerase III factors in the whole oocyte S150 extract.
The specific aims of this proposal are: 1. to determine which factor in the S150 extract preferentially interacts with the somatic 5S gene as a result of the sequence differences preceding the promoter. 2. to investigate why these sequence differences provide a transcriptional advantage in the oocyte S150 extract but not in nuclear extracts. 3. to more accurately determine which sequence differences are most significant to this effect. 4. to investigate why E190 and tDNAmet genes are differentially expressed in the S150 and nuclear extracts. 5. to investigate why E190 and tDNAmet do not compete with 5S DNA in the S150 but do compete in the nuclear extract. Do these findings reflect differences in the factor populations? 6. to investigate why 5S, E190, and tDNAmet have similar activities in a heterologous extract (HeLa), but radically different activities in the homologous extracts.
