Protein interactions: analysis using allele libraries.

Interaction defective alleles (IDAs) are alleles that contain mutations affecting their ability to interact with their wild type binding partners. The locations of the mutations may lead to the identification of protein interaction domains and interaction interfaces. IDAs may also distinguish different binding interfaces of multidomain proteins that are part of large complexes, thus shedding light on large protein structures that have yet to be determined.

A novel approach for generating full-length, high coverage allele libraries for the analysis of protein interactions.

The yeast reverse two-hybrid method was developed to identify mutations disrupting protein-protein interactions. Adoption of the method has been slow, in large part, due to the high frequency of truncation and frameshift mutants typically observed with current protocols. We have developed a new strategy, based on in vitro recombinational cloning and full-length selection in Escherichia coli, to eliminate this background and dramatically increase the efficiency of the reverse two-hybrid protocol.

C. elegans GLA-3 is a novel component of the MAP kinase MPK-1 signaling pathway required for germ cell survival.

During oocyte development in Caenorhabditis elegans, approximately half of all developing germ cells undergo apoptosis. While this process is evolutionarily conserved from worms to humans, the regulators of germ cell death are still largely unknown. In a genetic screen for novel genes involved in germline apoptosis in Caenorhabditis elegans, we identified and cloned gla-3.