Identification of proteins that interact with CeABF-1 using A yeast two-hybrid system

The helix-loop-helix (HLH) family of transcription regulatory proteins are fundamental regulators in the processes of cell proliferation and differentiation, cell lineage determination, myogenesis, neurogenesis, and sex determination in a wide range of multicellular organisms. A gene encoding a novel class II HLH protein has recently been identified from a human B-cell eDNA library using a yeast two-hybrid screen. The predicted human ABF -1 polypeptide sequence was used to search the Caenorhabditis elegans genome database for a C. elegans ABF-1 homolog. This bHLH protein, called C. elegans ABF -1 (CeABF -1 ), has a bHLH domain that shares 72% amino acid similarity with its human ABF-1 relative. The expression of the CeABF-1 mRNA has been detected in larval stages L2, L3, L4, and adult, however the mRNA is most highly expressed at the L3 and L4 stages. CeABF -1 protein is capable of heterodimerizing with the human E2A gene product, E4 7. Like human ABF -1, CeABF -1 expression in the presence of the E4 7 protein results in a reduction in E2A mediated gene activation. It has therefore been concluded that CeABF -1 , like human ABF -1 , also acts as a transcriptional repressor. Because C. elegans shares many conserved genes with higher eukaryotic organisms it has become a model organism for in depth genetic studies. It has therefore become increasingly desirable to investigate the possibility of alternative protein-protein interactions that can potentially occur within C. elegans, so it was necessary to construct a C. elegans eDNA library along with the appropriate bait vector expressing the CeABF- 1 protein. The titer ofthe primary library was calculated to be 9.7 x I06 clones, 10-fold greater than minimum titer requirement of I x I 06 clones for a good representational library. Sequencing of the CeABF -I insert confirmed successful construction of a mutation-free bait construct suitable for use in yeast two-hybrid screening. Yeast-two hybrid analysis revealed two new interactors, one of which was identified as an aldose reductase homolog, while the other remains uncharacterized