CLONING OF MIX-RELATED HOMEODOMAIN PROTEINS USING FAST RETRIEVAL OF GEL SHIFT ACTIVITIES, (FROGS), A TECHNIQUE FOR THE ISOLATION OF DNA-BINDING PROTEINS

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA, Using this technique, we have isolated two proteins that ar e structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expre ssed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in disti nct, yet overlapping regions in mesoderm and endoderm, The members of the Mix family heterodimerize with each other and overexpression of ea ch results in severe axial abnormalities, Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot, In jection of Mix,3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeopro teins are downstream effecters of growth factor signaling during endod erm and ventral mesoderm formation.