The general problem proposed for investigation is the analysis of mammalian dosage compensation. In particular the project will be concerned with the molecular analysis of the X-inactivation mode of dosage compensation used in humans, mice and a number of other mammalian forms. Chromosomal dosage control is critical to normal growth and development, and X-inactivation brings about the essential equivalence of X-chromosome dosage in the somatic cells of females and males. No euploid fetus or individual with two active X-chromosomes has been observed, suggesting that X-chromosome dosage imbalance is lethal. There are three stages in the X-inactivation process: X-inactivation in the extra embryonic lineage at blastocyst formation, X-inactivation in the embryonic lineage during the very late blastocyst stage and reactivation of the inactive X-chromosome in the germ cell lineage near the time of oogenesis. At the molecular level three sequential steps must be considered: initiation of inactivation at a single site or control center, spreading of inactivation along the chromosome, and maintenance of the inactivated state throughout the cells' somatic history. Through the use of various X-linked probes, in situ nick translation and high voltage electron microscopy of sex chromatin, we will investigate these different levels of developmental control of X-inactivation. Initial studies will concentrate on differences between the active and inactive X-chromosomes regarding specific X-linked genes, primarily the phosphoglycerate kinase (PGK) and the replication enhancer plasmid genes. X-linked cDNA and genomic probes will be used to look for chromatin conformation and DNA modification differences between the active and inactive X-chromosomes. Materials to be studied will include cell cultures varying in X-chromosome composition and murine tissues including extra-embryonic and embryonic yolk sac tissues. Chromatin configuration will be investigated using nucleases and DNA modification will be studied with restriction enzyme and sequence analyses. In situ nick translation and high voltage electron microscopy will be used to study sex chromatin and a possible nuclear envelope attachment site of this structure.
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