X inactivation, established during early mammalian development, results in the silencing of all but one X chromosome in cells of female embryos. However, genes that escape inactivation are interspersed on the X chromosome. The unusual expression of some of these genes may explain the association of Turner syndrome with the lack of one X chromosome. Our previous studies have uncovered major differences between human and mouse in terms of location and X-inactivation status of genes. This proposal exploits mouse systems to follow the timing of X inactivation and escape during development, to determine the X-inactivation status of genes in relation to their location and to examine evolution of dosage compensation. Specifically, we plan (l) to construct transgenic mice that contain reporter sequences inserted in the Hprt gene that is subject to X inactivation and the Smcx gene that escapes. Expression of the reporter sequences will be followed during development. We will also continue studies of a transgene that escapes X inactivation. Second, we plan (2) to construct transgenic mice with reporter sequences inserted at different locations from the inactivation center to follow spreading of X inactivation. A parallel approach will be to assay for allele-specific expression of X-linked genes by in situ hybridization. Third, we plan (3) to define the extent of a rearrangement previously detected by mapping of the Clcn4 gene to the X chromosome in one mouse species but to an autosome in another. Expression studies of Clcn4 will be done to determine the consequences of dosage compensation. Finally we plan (4) to determine the X-inactivation status of mouse genes using an X-autosome translocation system developed previously. This study in conjunction with detailed mapping will show whether genes that escape X inactivation are located in specific domains. The experiments planned based on expression and mapping studies of genes that escape X inactivation in comparison to those subject to inactivation, should further elucidate the processes of X-inactivation spreading and maintenance and of evolution of the sex chromosomes.
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