Molecular Analysis of Different Structural Forms of the rab-7 Gene

Our lab studies neural development in C. elegans, a transparent nematode used as a model system for gene function studies. Rab-7 is a gene in the rab family of monomeric GTPases responsible for vesicular traffic within the cell. Specifically, rab-7 regulates endosomal sorting and transportation of vesicles from early endosomes to lysosomes. In general, monomeric GTPases function as molecular switches. They alternate between active and inactive forms depending on their nucleotide binding state. When bound to GTP, the GTPase is active; when GTP is hydrolyzed to GDP it becomes inactive. Mutations in rab7 have been associated with patients suffering from Charcot-Marie-Tooth type 2B neuropathy. Studies into the function of rab-7 in C. elegans can give clues to the causes of this disease and leads into possible treatments in humans. We obtained cDNA entry clones from the ORFeome library (Reboul et al. 2003)and verified that the WT rab-7 cDNA sequence was correct. Using site-directed mutagenesis, we mutated specific codons in the wild-type rab-7 to create both a constitutively active (CA) and a dominant negative (DN) form. These three forms (WT, DN, and CA) will be recombined into a destination vector using gateway technology to create an expression clone that includes a neural promoter driving expression of rab-7 fused to mCHERRY. These expression clones will then be introduced into C. elegans to study rab-7 function in neurons. The mCherry fluorescent protein will show where the rab-7 protein is located within neurons. The DN and CA forms will help dissect rab-7 function.