Embryonal carcinoma (EC) cells have proven to be of tremendous value in analyses of both oncogenesis and mammalian development as well as in studies of the interrelatedness of these two processes. Of particular note are the use of these cells in the development of transgenic animals and in studies of retinoid mediated differentiation therapy. Previously we infected an EC cell line (NR1-0) with a defective retrovirus containing a neomycin resistance cassette (NeoR) and created a mutant cell line designated NR1-6. This cell line is unique in its morphological, adhesive, tumorigenic and differentiative properties. Genetic analyses of mutant, hybrid and revertant cell lines indicate that there is only a single retroviral insertion site regulating all of these recessive, and, apparently, genetically linked, phenotypess. To elucidate the molecular regulation of this pleiotropic mutation we have sequenced 17.1 kb of genomic DNA flanking the insertion site. With the exception of repeat elements, this region shares no significant homology with any previously reported sequence and is therefore a novel locus. Using a variety of techniques we searched for transcripts encoded both within this region and elsewhere in the genome whose expression might be directly or indirectly regulated by the insertion site locus. We have identified at least three novel exons encoded within the 17 kb flanking sequence, two of which hybridize to a transcript of 3.3 kb in northern analysis of mouse cells and tissues. Using differential display and mRNA fingerprinting we have also identified a number of potential downstream target genes whose expression differ between the NR1-6 and NR1-0 cell lines. We now propose to evaluate these findings in more detail in order to understand the molecular mechanisms regulating the original NR1-6 mutation and to identify the downstream acting loci which regulate the variant phenotypes (most particularly differentiative response to retinoids) associated with the NR1-6 mutation. To this end we will conduct further molecular and functional analyses of: the transcript(s) encoded by the insertion locus (Specific Aim 1), and transcripts encoded by other loci which have been shown to be differentially expressed between parent and mutant cells (Specific Aim2).