This chapter describes a genetic approach to investigate the nuclear order in yeast and discusses the development of a screen for mutations that disrupt nuclear organization. This approach relies on the observation that loop attachment complexes are evolutionarily conserved. The matrix associated region (MAR), initially characterized in the mouse κ-immunoglobulin gene, binds specifically to isolated yeast nuclear matrices. When inserted into a yeast promoter, the mouse κ-MAR interferes with transcription in vivo by interaction with yeast proteins. These observations were used to develop a genetic screen based on alleviation of the inhibitory effect of the MAR. This method is also applicable to the identification and/or characterization of other DNA/protein complexes. The method can be adopted to investigate the interaction between any DNA-binding protein and its recognition sequence, provided the protein is found in or can be expressed in yeast cells. The technique can also be used to clone DNA-binding proteins from mammalian cells. Yeast containing an assay plasmid with the relevant sequence inserted into the GAL1 promoter test site is transformed with the mammalian cDNA expression library and β-galactosidase activity of the transformants assayed on X-gal indicator plates.
ASJC Scopus subject areas
- Cell Biology