The histone genes coding for early and late embryonic histones in the sea urchin embryo have very different patterns of expression. The early genes, arranged in a tandemly repeated array, are expressed during stages until late blastula but are inactive at the gastrula stage. The late genes, organized irregularly in small clusters, although active to some extent in the early embryo, become highly active in the mid to late blastula stages. We have been studying the structure and regulation of these two gene sets and have demonstrated that their control is primarily at the transcriptional level. We have cloned and sequenced a late H3-H4 gene pair (SpL22) and traced the timing and extent of its transcription. In this proposal, we outline four major aims: 1) To isolate and characterize other late embryonic H3, H4 and H2A genes, some of which may have patterns of expression different from the SpL22 gene pair. 2) To investigate the timing, location, and extent of transcription of the late genes using nuclear run-on assays, in situ hybridization, and in vivo incorporation assays. 3) To determine the conformation of early and late histone genes in chromatin by nuclease sensitivity, exonuclease protection and footprinting, as well as to isolate the histone gene chromatin in its native state. 4) To identify and characterize the factors involved in the initiation of transcription, and sequences with which they interact, of both classes of genes. This part of the proposal is made possible by our recent development of an embryonic nuclear extract which accurately initiates both the early and late H4 genes. We will attempt to understand the differential regulation of the early and late genes by extract complementation, factor combination, pre-incubation and competition, and chromatin reconstitution experiments.
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