The human genome contains several ets-related genes (ETS1, ETS2, ERG, ERGB, ELK1, ELK2, SAP1, ELF, SPI1, SPI2). Other ets-related genes have been found in mammals (GABPalpha, PU.1/Spi-1, Fli-1, PEA3) and invertebrates (D-elg, E74), suggesting that additional human ets-related genes may yet exist. In addition to the carboxy terminal DNA-binding domain, a helix-loop-helix motif is highly conserved among ETS family members, suggesting it may play an important role in the function of ETS proteins. This motif has been postulated to be involved in protein- protein interactions. To understand the functions and mechanisms responsible for the functional differences between the ets-related genes, genes whose products are able to interact with ETS proteins will be identified. To achieve this goal, constructs containing defined segments of the open reading frames (ORF) from the human ETS1 and ETS2 genes have been placed into prokaryotic vectors that allow for overexpression and rapid purification of the fusion protein produced by each construct. Rapid purification is accomplished by high-affinity interaction between vector-derived histidine residues and nickel-chelated resins. Ets/vector constructs retain this affinity, affording a rapid means of obtaining Ets affinity columns. Proteins from labelled cell extracts which interact with these affinity columns can be identified. These vectors also contain defined protein cleavage sites that allow for isolation of the unfused protein. These proteins will be labelled and utilized as probes to identify gene products (and the appropriate genes) with which they are able to interact. This methodology will be applied to other genes and will allow us to begin to dissect the role(s) of the ETS genes in the complex network of gene regulation and cellular signal transduction.
