The proposed research extends ongoing studies which probe questions of chromatin and chromosome structure as they relate to chromosome function. In order to investigate questions of specific gene expression and processing, we have begun to develop in situ hybridization techniques at the electron microscope level. We are extending our initial success with autoradiography to non-autoradiographic detection. Biotin-substituted probes are hybridized to specimens on electron microscope grids and the hybrids detected by reaction with antibody against biotin, followed by second antibody coupled to one of several electron dense probes. We are also studying the composition of constitutive and facultative heterochromatin. We are analyzing proteins and DNA in centromere fractions derived from mouse metaphase chromosomes, and we hope to use a human cell line which possesses four inactive X chromosomes as a source of facultative heterochromatin. In order to assay for enrichment of this material, we have identified, cloned, and begun the characterization of a 2 Kb Bam Hl-generated fragment from the human genome which is concentrated at the centromere of the X chromosome. Further characterization of this sequence will be pursued and it will be utilized in attempts to purify human inactive X chromosomes. The general goal of these studies is a better understanding of the relationship between chromatin composition, organization, and gene function.