Grant-in-Aid for JSPS Fellows
|Allocation Type||Single-year Grants|
|Research Institution||Kyoto University|
Carlton Peter 京都大学, 生命科学研究科, 准教授 (20571813)
|Foreign Research Fellow
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
Suspended(Fiscal Year 2017)
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 2017 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2016 : ¥1,200,000 (Direct Cost : ¥1,200,000)
|Keywords||PPH-4 / immunoprecipitation (IP) / EMS mutagenesis / PPH-4.1 suppressors|
|Outline of Annual Research Achievements
The highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1 in C. elegans, is required to carry out several separate functions involving meiotic chromosome dynamics during meiotic prophase I, including programmed DNA double-strand break (DSB) initiation and crossover formation. In order to identify the interacting proteins of PPH-4.1, potentially including its substrates and opposing kinases:
(1)we performed immunoprecipitation (IP) with anti-FLAG antibodies to co-IP proteins that interact with PPH-4.1, using a 3x FLAG-tagged PPH-4.1 transgenic worm line. We did mass spectrometry analysis of eight enriched immunoprecipitated proteins. Unfortunately, none of them seem involved in meiosis process.
(2)We generated two transgenic lines, GFP-tagged PPH-4.1, aim to try anti-GFP antibodies to do co-IP. Unfortunately, these transgenic lines can’t rescue the pph-4.1(tm1598) null mutant.
(3)we generated two transgenic lines, BirA* tagged PPH-4.1, to perform proximity-dependent biotin identification (BioID) experiment which could detect proximal biotinylated proteins of BirA* tagged PPH-4.1. Unfortunately, these transgenic lines can’t rescue the pph-4.1(tm1598) null mutant.
The failed results of both (2) and (3) indicate fusion proteins are not functional or these single-copy transgene got silenced in the worm germline, due to RNA-induced epigenetic silencing.
In addition, we performed EMS mutagenesis to screen for PPH-4.1 suppressors, potentially including its opposing kinases. We have already got 73 candidate lines.
|Current Status of Research Progress
Current Status of Research Progress
2 : Research has progressed on the whole more than it was originally planned.
Since the conventional biochemical experiment-IP didn’t give us promising PPH-4.1 interacting proteins, we performed the genetical experiment- EMS mutagenesis to screen for PPH-4.1 suppressors.
EMS is the most commoly used mutagen in C. elegans and by far the most potent. EMS mutagenesis is biased towards G/C to A/T transitions, therefore, these mutations are typically strong loss-of-function or null allelles.
We used 50mM EMS to induce mutations in the DNA of sperm and oocytes of mixed population of pph-4.1 (tm1598) heterozygote/homozygote L4 larvae and young adults. Mutagenized worms were grown on agar plates for 2 generations to produce homozygous mutant strains. pph-4.1 (tm1598) homozygous mutants are non-GFP and sterile, therefore, the putative suppressor mutants with pph-4.1 (tm1598) in the background are supposed to be non-GFP and fertile. The F2 progeny were screened for non-GFP and fertile phenotype. Progeny of putative suppressor mutants were genotyped by PCR and confirmed they are pph-4.1 (tm1598) homozygotes. Overall, 10470 genomes were screened and 73 confirmed suppressors were isolated.
|Strategy for Future Research Activity
EMS mutagenesis could make multiple background mutations unrelated to PPH-4.1 functions in meiosis, therefore, first of all, we need to outcross pph-4.1 (tm1598); suppressor mutants against dpy-18 (e364) unc-64(e246)/+ males four times to remove background mutations.
After outcross, we are going to count the brood size and embryonic lethality of pph-4.1 (tm1598); suppressor mutants. Based on these results, we could roughly group these mutants, some of which could be robustly suppressing and others are only weakly suppressing. It is very possible to get multiple alleles of the same suppressor genes.
Our previous publication showed that autosomal pairing is diminished in pph-4.1 (tm1598) mutants, therefore, we could also look at the gonads by immunofluorescence, staining for ZIM-3 protein, which is specially required for homolog pairing, synapsis and segregation of chromosomes I and IV during meiosis. Our previous publication also showed that pph-4.1 (tm1598) mutants have seven or more DAPI-stained bodies in diakinesis oocytes, which indicate the failure of one or more chromosome pairs to undergo crossover formation. Accordingly, in the meanwhile, we could count the numbers of DAPI-stained bodies.
Based on all above results, we are going to send out some most interesting and promising lines for genome sequencing to characterize the suppressor genes.