卵母細胞における染色体ダイナミクス制御と母体の加齢効果

2017 Fiscal Year Annual Research Report

• Project/Area Number: 16F16088
• Research Institution: Kyoto University
• Principal Investigator: Carlton Peter 京都大学, 生命科学研究科, 准教授 (20571813)
• Co-Investigator(Kenkyū-buntansha): LI Xuan 京都大学, 生命科学研究科, 外国人特別研究員
• Project Period (FY): 2016-04-22 – 2019-03-31
• Keywords: EMS mutagenesis / PPH-4.1 suppressors

Outline of Annual Research Achievements

The conserved protein phosphatase PP4 homolog, PPH-4.1 in C. elegans, is required for meiotic chromosome dynamics. To identify its interacting proteins , we performed EMS mutagenesis to screen for PPH-4.1 suppressors. We have already isolated 73 candidate strains from the screen. After EMS mutagenesis, we need to outcross these 73 strains at least four times with wild type worms to clean up the background mutations unrelated to PPH-4.1 functions. Until now, 1 strain is outcrossed 6 times; two are outcrossed 5 times; six are outcrossed 4 times; one is outcrossed 3 times; three are outcrossed twice; three are outcrossed once. During outcross, according to the number of plates which showed embryonic lethal phenotype, the same phenotype as pph-4.1(tm1598) single mutant displays, we could estimate whether the suppressor genes are on Chr III or not, where pph-4.1 locates .To determine whether the suppressor strains restore the normal meiotic chromosome dynamics, including crossover formation, autosomal pairing, DNA double-strand break initiation and meiotic progression to pph-4.1(tm1598) mutant, we performed immunofluorescence experiment against ZIM-2, ZIM-3, and DSB-1 in suppressor strain #67. The results showed all of the pph-4.1(tm1598) meiotic defects previous described are partially rescued, except for the cell cycle delay. To determine the genetic properties of suppressors, we determined the percentage of viable pph-4.1(tm1598) embryos by strains heterozygous for each suppressor. 3 out of 71 strains’ genetic properties are determined, and all of them are dominant.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

One time of outcross takes around 2 weeks. Therefore, the progression of outcrosses are as expected. Our first strategy is to outcross pph-4.1 (tm1598); suppressor mutants against dpy-18 (e364) unc-64(e246)/+ males to remove background mutations introduced by EMS mutagenesis. The advantage of using dpy-18 (e364) unc-64(e246)/+ males are that by the Dumpy and Uncoordinated phenotypes, we can easily tell the worms are tm1598 homozygotes or heterozygotes, instead of doing multiple PCRs.
But we found this method takes longer time and is more frequently failed than outcrossing with the wild type N2 males. Therefore, later on, we have been using N2 males for outcross. The pph-4.1 (tm1598); suppressor mutants all showed reduced brood size and embryonic viability, therefore, by these phenotypes, we still can easily tell the worms are tm1598 homozygotes or heterozygotes, instead of doing multiple PCRs.
Outcross, immunofluorescence and counting the embryonic viability are routine work for all of the 73 strains we isolated, therefore, it is quite time consuming and laborious.

Strategy for Future Research Activity

Outcross, immunofluorescence (IF), and counting the embryonic viability and male percentage are routine work for all of the 73 strains we isolated, it is quite time consuming and laborious. In the past 6 months, the progression is as expected, but not highly efficient. In addition, for one suppressor strain #48, we already spent two months for outcross, but the further IF results showed individual difference varied a lot, therefore, it is not very worthy of further whole genome sequencing. We are planning to do a viability screen among these 73 strains. By roughly determining which strains have high viability, we could expect these suppressors strongly suppress pph-4.1. Based on their viability, we rank these 73 strains. I am going to focus on the highest ranking strains first.
After we get more strains outcrossed more than 4 times, we are going to send out them for whole genome sequencing to characterize the pph-4.1 suppressor genes.