1. A genetic screen identifies mutations in AtYAK1 that suppress lst8-1-1 growth defects
mutations in AtYAK1
- sol23, sol69 : suppressors the the lst8-1-1 mutant ← EMS
- atyak1-3, atyak1-4, atyak1-5 : loss-of-function mutations ←T-DNA
2. Atyak1 mutations suppress the delay in flowering and reduced hypocotyl growth observed in lst8-1 mutants
Atyak1 mutation : atyak1-4
A. flowered earlier
B. increased hypocotyl growth
3. Atyak1 acts in the TOR signaling pathway
A&B. Three atyak1 mutants(yat1-4, 1-3, 1-5) are less sensitive to AZD-8055 compared to WT
(resistant to AZD-8055)
C. Detection of an increase in TOR activity in the atyak1 mutants ← RPS6 immunoblotting
- TOR activity is reduced in the lst8-1-1 or lst8-1-2 mutants plants compared to WT
- no changes in TOR activity in atyak1 alleles
- AtYAK1 could be a downstream TOR effector
4. Atyak1 interacts with RAPTOR1B and is a substrate of TOR
A. Y2H -His AD-YAK1⇄BD-RPT
B. BiFC YFP nYFP-YAK1⇄cYFP-RPT
C. kinase assay
- when INDY-inhibited AtYAK1 recombinant protein was combined with the TOR fraction, phosphorylation of the AtYAK1 fusion protein was observed
5. The metabolic changes observed in lst8-1-1 are partly mediated by AtYAK1
A. amino acid increase occruing in lst8-1-1 mutants is reduced in lst8-1-1 atyak1-4 double mutants
B. the most striking increase was measured for glutamine
6. Mutations in Atyak1 partly reduce the gene expression changes observed in lst8-1-1 mutants
A&B. the loss of AtYAK1 activity globally dampens the impact of the lst8-1-1 mutation on the Aradidopsis transcriptome
C. gene ontology analysis
- the set of genes that were differentially regulated in the lst8-1-1 mutant and had their expression tempered or reserved in the lst8-1-1 atyak1-4 double mutant → diverse biologica processes
D. a significant overlap between ABA treatment and lst8-1-1 mutation
E. de-regulation was alleviated in lst8-1-1 atyak1-4 when compared to lst8-1-1
7. Atyak1 mutations suppress the ABA hypersensitivity of the lst8-1-1 mutant
A. seedlings grown in vitro on half MS medium
B. germination, cotyledon establishment
유전적 검사법(genetic screen)
돌연변이를 일으킨 집단에서 관심이 있는 표현형을 가진 개체를 식별하고 선별해 내는 사용하는 실험적인 기술로
유전자 기능뿐 만 아니라 분자단위의 생물학적 과정이나 경로에 대한 중요한 정보를 제공해 준다.
기본 검사법(Basic screening)
forward genetic screen : 개체의 특별한 표현형을 책임지는 유전자 또는 유전자 집단을 알고자 할 때 쓰이는 접근법
reverse genetic screen : 알고 있는 유전자를 파괴한 이후 개체의 표현형을 분석하는 방법
→유전자의 기능을 알아내기 위함
변형 검사법들(Screening variations)
suppressor screen : 원래의 돌연변이 표현형을 완화하거나 원래로 되돌리는 억제 돌연변이(suppressor mutation)를 확인하고자 할 때 쓰이는 방법
note! 원래의 돌연변이의 표현형을 억제시키는 동시에 연구하는 돌연변이와는 구별되는 염색체 장소에 일으키는 두 번째 돌연변이로 설명된다. 만약 돌연변이가 원래 돌연변이와 같은 유전자에 있다면 이는 유전자 내 억제로 불리고, 반면에 돌연변이가 다른 유전자에 위치하면 유전자 외 억제 또는 유전자 간 억제로 불린다.
EMS(Ethyl methanesulfonate) : produces random mutations in genetic material by nucleotide substitution
transition : a point mutation that changes a purine nucleotide to another purine(A⇄G), or a pyrimidine nucleotide to another pyrimidine(C⇄T)
nonsense mutation : アミノ酸コドン を終止コドン に変える変異
splice-defect mutation at the end of the first intron affecting the acceptor site(AG) was present
