Formstecher, Etienne, Tafelmeyer, Petra, Masson, Maryline, Pintard, Lionel, Boulin, Thomas, Bessereau, Jean-Louis
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Yeast two-hybrid (Y2H) protein interaction screening has proven instrumental for the analysis of the C. elegans interactome, thanks to dedicated resources such as ORF collections and oligo dT-primed cDNA libraries. 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 rates. To circumvent these limitations, we have used a domain-based strategy to construct two highly complex, random-primed C. elegans 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 in yeast, 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 score. 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.
[
European Worm Meeting,
2006]
Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.