Testing GLD-1 and SEL-10 Interaction Using the Yeast Two-Hybrid System

GLD-1 and SEL-10 are two proteins important for C. elegans germ line development. GLD-1 (defective in germ line development) contains an RNA binding domain (KH-type) and is essential for multiple aspects of germ line development including oogenesis. GLD-1 immunostaining shows that its protein expression is at its highest during meiosis, and abruptly decreases immediately prior to oogenesis. SEL-10 (Suppressor/Enhancer of Lin-12) consists of an F-Box domain and WD-repeat domain, and is involved in cell fate determination and ubiquitin-mediated degradation. This project focuses on the possible interaction between GLD-1 and SEL-10 proteins. Because SEL-10 is likely to partake in protein degradation, and a null mutation of sel-10 results in mis-expression of gld-1, we hypothesized that SEL-10 is involved in degrading GLD-1 during oocyte formation through their interaction, which would account for the disappearance of GLD-1 during later stages of oogenesis.

To test for the GLD-1 and SEL-10 interaction, we are using the ProQuest Yeast Two-Hybrid System from Invitrogen. In this system, one protein of interest, SEL-10 in our case, is attached to a protein known as DNA-Binding Domain (DB). The second protein of interest, GLD-1, is similarly fused to an Activation Domain (AD). The interaction of the DB and AD is required for the transcription of chromosomally-integrated reporter genes. If GLD-1 and SEL-10 do interact, their interaction will bring DB and AD into proximity that allows for transcription of the reporter genes. Then by culturing transfected yeast under selective conditions, it is possible to determine if GLD-1 and SEL-10 interaction exists. We are also interested in the specific regions of the SEL-10 and GLD-1 interaction. GLD-1 and SEL-10 are therefore cut into different-sized fragments ranging from approximately 70 to 400 amino acids. These constructs were designed to include moderate overlap between each fragment, and the functions of different parts of each protein, such as the F-box in SEL-10 and KH domain in GLD-1, were also considered during the design process.           

Thus far, we have done all the preparation needed for transfecting the yeast and testing for protein interaction. The yeast strain MaV203 from ProQuest was validated and tested for transfection efficiency. Also tested were five ProQuest control strains for their relative interaction strength and, later on in the project, for comparing with the GLD-1 and SEL-10 transfected yeast. Most of the constructs of GLD-1 and SEL-10 have been made using PCR, and are ready for insertion. Our next step would be to finish making all the constructs and begin testing one against another for their interaction.