[
International Worm Meeting,
2007]
Yeast two-hybrid (Y2H) protein interaction screening has proven instrumental for the analysis of C. elegans interactome, thanks to dedicated resources such as ORF collections and oligo dT-primed cDNA libraries1,2. However, map completeness has been limited so far by the use of full-length proteins (ORFeome) or C-terminal polypeptide fragments (oligo dT-primed cDNA libraries) that resulted in significant false negative rates3. To circumvent these limitations, we have used a domain-based strategy to construct two highly complex, random-primed cDNA libraries. The first library has been constructed by combining equimolar amounts of mRNA from 4 different N2 samples to increase transcript diversity: (1) males and hermaphrodites (all stages including embryos), (2) starved mixed stage culture, (3) heat-shocked mixed-stage culture, (4) dauer stage. The second library was prepared exclusively from C. elegans embryos of all stages. The complexity of both libraries is greater than 10 million independent fragments, with an average fragment size of 800 bp. To ensure reproducible and exhaustive Y2H results, these libraries are screened at saturation using an optimized mating procedure. This allows to test on average 100 million interactions per screen, corresponding to a 10-fold coverage of the library. As a consequence, multiple, independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score4. These two C. elegans libraries have been integrated into our high-throughput yeast two-hybrid platform and are available for screening on a fee-for-service basis. The results of representative screens performed on both libraries will be presented at the meeting. Please send inquiries to: Etienne Formstecher, eformstecher@hybrigenics.com 1. Zhang et al., 2004, Nature Genetics, 36:507 2. Li et al., 2004, Science, 303:540 3. Han et al., 2005, Nature Biotechnology, 23:839 4. Formstecher et al., 2005, Genome Research, 15:376.