The role of cytochrome P450 enzymes in caenorhabditis elegans dauer recovery

Abstract

The purpose of this experiment is to determine if cytochromeP450 (cyp450) enzymes play a role in dauerrecovery of Caenorhabditiselegans.The life cycle of this free-living nematode is divided into several larval stages. Under stressful conditions, L2 larvae molt into the dauerstage, an alternate life cycle stage designed to cope with undesirable conditions. The dauerstage may be analogous to infective stage parasitic nematodes, some of which cause human disease. Microarray analysis has identified four cyp450genes that are expressed transiently during dauerrecovery. We are using double stranded RNA interference (dsRNAi) to knock out these genes. Using this technique in silencing the genes of interest, we will be able to observe a null phenotype in comparison to that of normal gene function in worms recovering from dauer. A cyp-14A5dsRNAiplasmid was purchased (GeneserviceLtd.), but gene specific dsRNAiplasmids were not available for the other three genes. We have amplified these three cyp450genes (cyp-13A4, cyp-13A5and cyp-13A10)and have cloned the genes into E. coliplasmids for use in the dsRNAiexperiments. Using DNA sequencing, we have thus far confirmed that that the cyp-13A10plasmid is correct, however we are in the process of resequencingthe other two plasmids. Initial results have shown delayed recovery from dauer, however, we are repeating the dsRNAiexperiments and will report the results. We predict that a defect will be observed in the ability of the worms to recover from daueras compared to the controls