The long range goals of the experiments outlined in this proposal are to understand at the molecular and biochemical level what determines temporal and spatial patterns of gene expression during early embryogenesis. It has been made clear from numerous studies that cells committed to different lineages differ in the patterns of genes they express. A fundamental question concerning developmental biologists is just how these different patterns are generated in the daughters of a single cell, the fertilized egg. Our approach to this problem is to dissect the cis-acting regulatory sequences and the trans-acting regulatory proteins of families of genes encoding histone H1 proteins that are differentially regulated during early embryogenesis and in adult tissues of the sea urchin. The expression of the early or embryonic histone genes, which are encoded by 300-500 tandem arrays, is confined to a period up to the blastula stage of development about 12 hrs. following fertilization. The late histone H1 gene family consists of 2 single copy genes whose transcripts are expressed at low basal levels until the blastula stage when their transcription rate increases. These late genes are used in all adult tissues of the animal with the exception of male germ cells in testes. Finally, we will study a testis specific H1 gene. Our experimental approach to the questions outlined above has been to identify the DNA sequences required for the accurate initiation of transcription and then to correlate them with sequences necessary for correct temporal expression. We will test various DNA constructs altered by site-specific mutagenesis in microinjected sea urchin zygotes and in in vitro transcription systems. We will purify and characterize the proteins that bind to each of the important cis-acting regulatory sites and we will test their biological activity, produce antisera, and determine when and where these proteins are present in oocytes, eggs, embryos and adult tissues. Finally, we will isolate and characterize cDNAs encoding some of these regulatory proteins. These studies pertain directly to understanding the precise and detailed molecular mechanisms underlying differential gene expression during embryogenesis and differentiation.
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