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The puromycin-sensitive aminopeptidase, PAM-1, is crucial for polarity establishment and asymmetric division, which are necessary for viable development of one cell C. elegans embryos. We have found restorative effects of these phenotypes in pam-1 worms by adding a secondary suppressor mutation in wee-1.3 (lz5). WEE-1.3 is an inhibitory kinase that regulates maturation promoting factor (MPF) to control the timing of oocytes entering M phase during interphase of mitosis. This allows oocytes to mature at a controlled rate before fertilization occurs and meiosis concludes. To investigate additional interactions between PAM-1 and WEE-1.3, we observed how the knockout of wee-1.3 using RNA interference in wildtype and pam-1 worms affected oocyte maturation. Wildtype worms that were grown on wee-1.3 RNAi plates became sterile. This is due to oocytes maturing precociously and attempting to undergo meiosis before fertilization, which we were able to visualize by scoring the maturity markers of nucleoli and phosphorylated histone presence. In contrast, pam-1 worms that were grown on wee-1.3 RNAi plates were not sterile and their nucleoli counts seemed to revert back to what we would expect to see in wildtype worms, however we need to collect more data on phosphohistone staining to draw any conclusions. We suspect that pam-1 and wee-1.3 interact during oocyte maturation in addition to early embryogenesis. We used meiosis and meiotic exit timing, the time between the extrusion of the second polar body and the formation of the pronucleus, to see if wee-1.3, could rescue early meiosis in pam-1 mutants. We found that there was no significant difference between the duration of meiosis in pam-1 mutants and wildtype, however meiotic exit timing was delayed but we were unable to complete our analysis. To test if PAM-1 may regulate the levels of WEE-1.3, we measured the intensity of WEE-1.3::GFP levels in wild-type and pam-1 mutants. While more data is needed to draw conclusion, we saw a trend that pam-1 embryos have higher levels of WEE-1.3, suggesting that WEE-1.3 may be a target of PAM-1. Therefore, we have evidence of multiple interactions between PAM-1 and WEE-1.3 during the development of a C. elegans embryo.
Eisele, Caprice, "Does Using wee-1.3 as a Secondary Suppressor Restore Wildtype Timing of Meiosis and Oocyte Maturation in pam-1 C. elegans Mutants?" (2020). Biology Presentations. 9.
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