The proposed research is to isolate sequences from the human genome which are involved in commitment to fat cell differentiation. The primary structure of such sequences will be determined and their genetic regulation and molecular interactions during adipogenesis and cell growth will be explored. To accomplish this, high molecular weight DNA from human fat tissue biopsies will be used in transfection assays to transfer the commitment genes to uncommitted mouse fibroblasts. After an additional round of transfection to reduce human complexity in the recipient mouse cells, the DNA from the secondary transfectant clones which fat-convert under appropriate conditions will be used to construct libraries. Committing sequences will be isolated from such libraries by hybridization to human specific probes. The isolated genes will be sequenced and used to define structural DNA changes in uncommitted, committed, and fully differentiated adipocyte cells. Molecular interactions of the isolated sequences with upstream regulatory elements of adipocyte specific proteins will be defined by biochemical means. The gene(s) will be mapped in the human and murine genomes. %%% The findings from such a study will bring us close to an understanding of the genetic regulation of fat cell differentiation. A cloned sequence involved in commitment to fat cell differentiation will help in a broad range of studies dealing with the molecular basis of human diseases such as obesity and heart disease, where the normal processes of cellular growth control, hormonal regulation and differentiation may have been subverted.
