DESCRIPTION: Innovative interdisciplinary work is essential to address the most challenging issues in the era of genomic biomedical research. One of these is the evolutionary structure/function of transcriptional regulation. Comparative molecular evolutionary work is essential here. TP53 is probably the single most important gene in cell cycle and cancer studies. However, there is no clear model for the evolution of the main transcriptional regulatory region across mammals, nor how our key animal models, including mouse, differ. Going from a shrew-like ancestor to an elephant, a whale or a human implies important changes in the regulation of TP53. While many mammals retain a similar TP53 cis-regulatory region to humans, evidence of its overall importance, others have undergone large changes, e.g., mice are highly atypical and one close relative of primates has deleted nearly the whole 5' regulatory region. This project's aims are as follows: (1) Very dense comparative alignment of the TP53/GBN5 cis-regulatory region across mammals; (2) Characterization of transcriptional responses using cell lines from a wide variety of species; (3) Molecular footprinting of this promoter region across mammals; (4) Test hypotheses of regulatory evolution using deletion/site-directed mutation constructs with reporter gene assays. As with (2) a key novel aspect of this work is the use of wildtype fibroblast cell lines from many different orders of mammals; (5) Using a novel pair-wise cell line assay, separate cis (local) from trans (non-local) effects in the evolution of transcriptional regulation; (6) Use, test, and develop bioinformatic/genomic and molecular evolutionary techniques on the dense sequence alignment of (1), plus genomes of over 14 species of mammals. To compare and contrast their effectiveness with our direct molecular data, Overall, to arrive at a better understanding of this important regulatory region and to illustrate the enhanced potential of comparative bioinformatics/genomics plus comparative functional molecular biology.