The investigator propose to study the effects of developmental regulation upon the protein composition and chromatin structure of specific eukaryotic genes. A novel isolation technique, nucleoprotein hybridization, enables the enrichment and subsequent study of chromatin fragments of specific genes. The first step in the procedure is the solubilization of chromatin from nuclei with a restriction enzyme. 5' exonuclease digestion yields chromatin fragments with single-stranded termini about 75 bases in length. An oligonucleotide is synthesized that is complementary to the exposed end of the gene of interest. This hybridization probe carries a biotin affinity label at its 5' end. Solution hybridization between the probe and chromatin is performed at 37 degrees c in 50-100 mM monovalent salt. Immobilization of the hybrids is achieved on an Avidin D affinity matrix. Extensive rinsing is followed by specific elution with DTT which cleaves a disulfide bond located in the linker. A fraction highly enriched in the genes of interest is obtained. All steps are carried out under conditions known to preserve chromatin structure and histone composition. The investigator will study the chromosomal proteins of the developmentally-regulated sea urchin early histone gene repeat (SUEHGR) from S. purpuratus. These five genes undergo a coordinated switch in expression during embryonic development. The genes have been enriched to 89 percent purity without noticeable nucleosome loss or rearrangements, or degradation of DNA or protein. Biochemical and physical studies of the transcriptionally active and inactive genes will determine the changes in gene structure that occur during embryonic development. The sequence variants and post-translational modifications of the histone will be mapped along the genes at different stages of development, using two dimensional gel electrophoresis of 125-I labeled samples. This will be the direct study of the proteins naturally bound to a specific gene. It will elucidate the mechanism of eukaryotic gene regulation during embryogenesis.
