Background Elongin C (Elc) is a highly conserved protein that forms several multiprotein complexes in various eukaryotic cells, including humans and yeast. Elc is overexpressed in a number of cancer cell lines. Elc forms a complex with the von Hippel-Lindau tumor suppressor protein (VHLp). Without Elc, VHLp is degraded and is unable to perform its function. Mutations in VHLp, some of which stop interaction with Elc, may predispose individuals to certain cancers. A region of VHLp, the BC box, is necessary for interaction with Elc. It is mutated in several cancers. BC box-like regions exist in other proteins, including ASXL-1, a regulator of transcription. Dr. Hyman's lab found that ASXL-1, in a mammary cDNA library, interacts with Elc. I hypothesized that mutating the BC box of ASXL-1 would interfere with the interaction.
Study Design and Methods ASXL-1 gene was mutated through site-directed mutagenesis changing the cysteine at position 1,508 to glycine and adding a new restriction enzyme recognition sequence (to identify mutants). The mutated plasmid was transformed into yeast cells as was the gene for wild-type ASXL-1. The two strains were subjected to the yeast two-hybrid screen, which originally identified interaction of ASXL-1 with Elc. Growth of the yeast on appropriate selective media indicates interaction. Growth was compared side by side.
Results The yeast with the mutant plasmid and the yeast with the wild-type plasmid grew equally well.
Conclusion These results indicate that there is no difference in the level of interaction between mutant ASXL-1 and Elc and the level of interaction between wild-type ASXL-1 and Elc. The cysteine may not make a critical contact or the proteins may be interacting through another site altogether.