| 研究実績の概要 |
Our attempts to create functional biotin ligase (BirA) fusions to PPH-4.1 protein were not successful. Creation of any fusion protein to PPH-4.1 has in fact been difficult, with most fusions leading to loss of essential catalytic activity. Thus, direct binding of PPH-4.1 to targets has not been possible to carry out in the current term.
In contrast, we have successfully carried out a genetic suppressor screen (as planned in our proposal in case the BirA fusions did not work). Using EMS mutagenesis, we have obtained 70 candidate suppressor lines that show increased viability in a homogyzous pph-4.1 deletion background. After removing extraneous mutations by back-crossing to pph-4.1 mutants, and carrying out whole-genome sequencing, we have mapped the suppressor mutations to small chromosome regions in 4 of these lines. We are currently testing candidates from within these regions for interaction with pph-4.1 by RNAi, double-mutant analysis, and CRISPR-induced recreation of candidate mutations. The activities of these suppressors will be important to dissect the multiple independent functions of PPH-4.1 in meiotic prophase. Importantly, the fact that suppressors were recovered at all demonstrates that genetic analysis of PPH-4.1 is a tractable approach.
Overall, this funding has led to our uncovering new, critical aspects of phosphoregulation in meiosis and has enabled a robust ongoing research program.
|
