east two transgenic lines were generated for each co-localization experiment. We produced transgenic lines carrying pRP2232 with gpa-15::gfp and pMH86 as marker plasmids. Males from these transgenic Comp Funct Genom 2003; 4: 479491. 482 E. Cuppen et al. lines were crossed with transgenic lines carrying pRP2229 or pRP2230 to give rise to transgenic animals that express both gpa-15::gfp and myo3::gfp::gpa-7WT or myo-3::gfp::gpa7QL. GFP expression was observed under a Zeiss Axiskop 2 equipped with a fluorescent light source and Nomarski optics. Laser scanning confocal microscopy was 
used to visualize GFP expression at the plasma membrane. Results cDNA cloning of all C. elegans G subunits C. elegans G subunit cDNAs were cloned based upon gene predictions and EST data present in ACeDB. When RTPCRs failed, predictions were re-examined using three-frame translations of genomic sequence in ACeDB, compared with a multiple protein sequence alignment of the known C. elegans, mouse and human G subunits. We cloned the cDNAs for all G subunits, except for GPA-9, for which we were not able to amplify the 5 half of the gene, and GPA17, which was discovered only recently. Sequence analysis shows that about 12% of the intronexon boundaries, including the manually corrected start and stop codons, were not correctly predicted by GENEFINDER and annotated in ACeDB. Furthermore, no evidence for alternatively spliced isoforms was found. Yeast two-hybrid interaction screen Each of the cloned C. elegans G subunits and constitutively active GTPase-deficient mutants of GPA-5, GPA-7, GPA-12 and GSA-1 were used as bait in a yeast two-hybrid interaction screen. 815 colonies grew on HTLA- plates, of which 547 turned blue on X-gal. 537 prey plasmids were successfully rescued and used to confirm the interaction specificity in a mating assay with the original and control baits. 100 interactions could not be reproduced and 137 were unspecific. The inserts of the 300 remaining clones were sequenced and compared with database sequences. Based upon the molecular identity, 103 clones were excluded from further analysis, as they encode GW 501516 web proteins that reside in cellular compartments such as the mitochondrion, or are extracellular, these Copyright 2003 John Wiley & Sons, Ltd. being locations in which G proteins are not expected to be functional. Furthermore, a set of common false-positives, the heat shock proteins, was discarded. The remaining 197 candidates were tested in a large yeast mating matrix for their interaction characteristics with all 19 cloned G subunits. In this experiment, additional control baits were included. At this point, we found that the original GPA-1 bait contained a frameshift mutation in the middle of the cDNA insert, resulting from a RTPCR cloning error. As lin-36 has a synthetic multivulva phenotype when combined with mutations in class A synMuv genes, we generated double mutants between goa-1 and lin-36 or lin8. However, no synMuv phenotype was observed. Although there is only a single homologue for Copyright 2003 John Wiley & Sons, Ltd. nucb-1 in C. elegans, whereas there are two in mammals, mutants behave completely like wildtype animals and no modulation of goa-1 lossor gain-of-function mutant phenotypes could be observed, suggesting a non-essential role for this protein in C. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19822652 elegans. Similarly, for a genetic mutant of nhr-22, no behavioural, developmental or male-mating defects were observed, nor modulation of gpa-7 loss- or gain-of-function muta