The long range goal is to understand the mechanism of control of genic expression on the human X-chromosome i.e. the single active X phenomenon. This phenomenon provides and especially important opportuinty to study a gene control mechanism that may be used ubiquitously in the human genome. We will attempt to derepress the HPRT locus and other loci on the human inactive X in pre-DX mouse x human hybrid cells with mutagens in order to mark controlling nucleotide sequences with mutations. Relevant DNA from such """"""""DX-hybrids"""""""" will be cloned and compared with DNA from cells in which the genes remain repressed. Other clones of X-chromosomal DNA will be prepared by making cDNA from DX hybrid mRNA that has been enriched for transcripts of derepressed genes. The cDNA clones will be used to isolate the corresponding genomic DNA clones. Cloned DNA of active and inactive X's will be compared by restriction digest analysis, nucleotide sequencing and other means in order to discover differences in sequence base, modification or structure.
