The general objective of the proposed research is to identify and characterize genes which regulate cellular proliferation, lineage determination and differentiation during nephron formation in the human kidney. The molecular cloning of such regulatory genes will likely identify molecular mechanisms required for normal renal development. This research is therefore directly relevant to the study of congenital and heritable renal malformations in humans, and holds promise for the identification of genes which regulate cellular proliferation in the kidney following toxic or immunological injury. Genetic and Biochemical approaches will be used to accomplish the following aims: 1. Establishment of the temporal and spatial pattern of expression of the POU class of Homeodomain proteins in the human kidney during development- Methods to be employed in this study include the electrophoretic mobility shift assay for the detection of sequence specific DNA binding proteins, Polymerase chain reaction (PCR) detection of specific mRNA's in microdissected tissue samples, and immunocytochemistry using specific antisera for the detection of members of the POU gene family. 2. Molecular cloning of the POU gene family expressed during renal development- Preliminary results in this proposal demonstrate the presence of a novel Octamer binding protein of the POU family expressed in the human kidney at the time of active nephron formation. A major goal of this research is to clone the gene encoding this protein. Molecular cloning of all POU genes in the kidney is also to be undertaken using degenerate primer PCR technology. 3. Biochemical Structure/Function studies of POU proteins- Novel POU genes identified by molecular cloning approaches will be characterized in vitro and in vivo to map protein domains involved in transcriptional activation, DNA binding and protein-protein interactions.
