Ingo Buessing et al. (2003) International Worm Meeting "Functional analysis of the single calmodulin and the four calmodulin like genes of C. elegans by RNA interference and 4D microscopy"

Ralf Schnabel, Ekkehard Schulze, Ingo Buessing, Jian Wang, Anton Karabinos, Klaus Weber

[International Worm Meeting, 2003]

Calmodulin (CaM), a small calcium binding protein, is a key mediator of numerous calcium induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Little is known about the functions of CaM-like genes. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we have used RNAi to analyse the functional importance of the single calmodulin gene (cmd-1) and the four calmodulin-like genes (cal-1 to cal-4) present in C. elegans. We find that RNAi leads to embryonic lethality for the calmodulin gene cmd-1 and an uncoordinated phenotype for the cal-4 gene. By detailed analysis of the cmd-1 RNAi embryos with the 4D-Microscope system we observed disturbed morphogenesis, aberrant cell migration patterns and a striking hyperproliferation of cells in the treated embryos. Normal apoptosis is suppressed in some instances, but in addition ectopic cell deaths occur and engulfment of cell corpses is inhibited. RNAi also allows several genes to be inactivated simultaneously. The triple RNAi approach for the cal-1, cal-2 and cal-3 genes gives rise to a complex phenotype while individual inactivations do not lead to a scorable phenotype.

Valenzuela MRL et al. (1991) International C. elegans Meeting "STRUCTURES AND FUNCTIONS OF THE REPEATING MOTIFS IN TWITCHIN."

Valenzuela MRL, Benian GM

[International C. elegans Meeting, 1991]

Twitchin, the 753,570 Da polypeptide encoded by the unc-22 gene, was the first member of a growing family of intracellular and mostly muscle proteins, found in diverse animals, composed of multiple copies of motif I (fibronectin type III domain-like) and motif II ( immunoglobulin C2 domain-like). To date, this family includes, smooth muscle and non-muscle myosin light chain kinases, titin, C-protein, 86 kDa protein, skelemin and probably insect projectin. The similarities to motifs found in extracellular and cell surface proteins engaged in recognition or adhesion suggests that motifs I and II of muscle proteins are also involved in binding--probably to myosin, other thick filament components, and also binding of these proteins to themselves and other family members. We hope to be able to demonstrate binding of small numbers of these motifs to thick filament components both in vitro and in vivo. Crystallographic structure determination will be done in collaboration with Pamela Bjorkman (Cal Tech). Using the pGEX- 2T vector, we have been able to express and purify large quantities of motif I, motif II and the predominant triplet I,I,II as glutathione-S- transferase (GST) fusion proteins in E. coli. We have succeeded with the thrombin cleavage of the motifs from GST and are trying ELISA-type binding assays with nematode and rabbit myosin. For in vivo 'binding' studies, we have cloned motif I, motif II, the trio I,I,II and the C- terminal 5 motif IIs into Andy Fire's pPD30.38 vector which directs body wall muscle expression. These constructs will be microinjected into unc-22 nulls and transgenics examined by immunofluorescence for co-localization to myosin.

Brigitte L LeBoeuf et al. (2004) West Coast Worm Meeting "Identifying and Characterizing a Mutation Involved in Male Mating Behavior"

L Rene Garcia, Brigitte L LeBoeuf

[West Coast Worm Meeting, 2004]

C. elegans males have a set of stereotypical mating behaviors that can be used to dissect the genetic and molecular pathways involved in behavior. Males mate by locating and positioning their spicules over the hermaphrodite's vulva, inserting their spicules, and then transferring sperm. To study how spicule insertion behavior is regulated, we isolated 5 mutations: sy557, sy558, sy559, sy574, and rg2 that cause the male's spicules to protrude from their bodies in the absence of mating cues. sy557 is located in unc-103 , whereas the other 4 affected genes have not been identified. UNC-103 is an erg-like K+ channel that is postulated to control male mating behavior. UNC-103 is expressed in many neurons in the head, ventral cord, and male tail, but sy557 seems to only affect spicule protraction. sy557 causes 66% of males to display the Pr otraction c onstitutive (Prc) phenotype. To understand how unc-103 interacts with other genes that control the timing of spicule protraction, we made double mutant combinations between unc-103(sy557) and the other mutations. unc-103(sy557); sy558 and unc-103(sy557); sy559 males are 100% Prc, suggesting that these combinations of mutations lead to a deficit in behavioral control that is too difficult to overcome. In contrast, the unc-103(sy557); sy574 double mutant males are 78% Prc, similar to the 66% of sy557 and 54% of sy574 . Since unc-103 might function in the same pathway as the gene affected by sy574 , we are identifying the gene that is affected by sy574 to understand how unc-103 may regulate behavior. sy574 maps to a 570 kb region on LG IV between unc-24 and dpy-20 . We have narrowed the region to between SNPs K08F4 and R102. Candidate genes that are located in this region include unc-43, gpa-7, tax-6, and cal-4 . Mutations in unc-43, gpa-7, and tax-6 complement sy574 , indicating that the mutation is not in these genes.