U7 is a small RNA previously shown of function in the 3' processing of histone mRNAs in man, mouse, and sea urchin. Although several studies have focused on the mechanism of U7's participation in histone synthesis, no investigation has yet focused on a potential link between the expression of U7 and histone genes. We propose to exploit the wealth of information concerning the naturally synchronous cell cycle of physarum macroplasmodia, to ideal suitability of the macroplasmodia for microinjection, and our finding that Physarum U7 and histone mRNA accumulation may be coordinated, to provide a powerful in vivo system for investigation U7's function. The goals of this AREA application are to: 1) identify and characterize the bona fide """"""""functional"""""""" Physarum U7, 2) construct a probe for this RNA, 3) identify potential differentiation-specific U7's, and 4) assay the accumulation of U7 throughout the cell cycle. Specifically, the characterization of a candidate Physarum U7 will be completed, and the cnadidated will be assayed for histone mRNA processing activity using both heterologous and homologous microinjection assays. If necessary, other candidate U7's will be identified using a DNA oligomer approach. Once bona fide U7 has been positively identified and characterized, a probe will be devised for this molecule. Using this probe potential differentiation-specific U7's will be identified by Northern analysis. The probe, and a cloned Physarum H4 gene provided by another laboratory , will also be used in hybridization analyses to assay the abundance of U7 and H4 mRNA throughout the synchronous cell cycle. If the initial findings of a coordinate expression hold true, we will be poised of investigate the mechanisms responsible for the periodicities. In the long range, the ability of Physarum plasmodia to fuse, resulting in cell cycle adjustments, could be exploited to assay U7/H4 mRNA coupling. It could be determined whether there is an actual coupling of U7 and H4 gene: 1) replication-timing, 2) replication-transcription, or 3) transcription-timing. The U7 gene and flanking regions could be characterized to determine whether any similarity exists with the known flanking regions of the Physarum H4 histone gene. Eventually, the Physarum system could provide a much better understanding of U7's role in histone synthesis than could ever be obtained from standard in vitro assays.
| Adams, D S; Griffin, L A; Nachajko, W R et al. (1991) A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor. J Biol Chem 266:8476-82 |
