The objective of these studies is to understand the mechanism regulating changes in the stability of histone mRNA during the cell cycle in cultured human cells. Histone mRNA rapidly accumulates in cells following the onset of DNA replication but is turned over rapidly at the end of S-phase or in the presence of inhibitors of DNA replication. Experiments in this proposal are designed to identify specific regions in the primary sequence of a histone H4 mRNA that are involved in cell cycle and DNA synthesis dependent post-transcriptional regulation. Our approach will involve fusion of a cloned H4 structural gene with regulatory elements from a human heat shock protein gene. Upon introduction of a plasmid containing this fusion gene into mammalian cells, expression of the histone gene can be induced by a shift in temperature independent of DNA replication. The heat-inducible histone gene will contain a marker sequence allowing detection and quantitation of transcripts in the presence of endogenous histone mRNA. A series of deletions will be made within the histone structural gene region by in vitro mutagenesis. Using in vivo gene expression assays in COS cells and HeLa cells, plasmids containing various deletions will be screened for histone transcripts which are stable when DNA synthesis is inhibited. Deletions which results in altered H4 histone mRNA stability will be introduced into stably transformed cell lines. We will then determine whether the same deletions abolish the relationship between DNA replication and histone mRNA stability during the cell cycle using synchronized cells. By comparing the sequence of deletion mutants which produce transcripts with altered stability with the sequence of the original gene, regions of the mRNA involved in post-transcriptional control will be delineated. These studies should give insight into one of the basic mechanisms by which cells coordinate the synthesis of major components of the chromosome during chromosome replication and cell proliferation.
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