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Summer researchSummer research
研究主題:研究主題: Yeast One Hybrid systemYeast One Hybrid system
中央研究院生農所籌備處 楊文中央研究院生農所籌備處 楊文欽教授欽教授
生科 04 級 881714 何國維
Institute introductionInstitute introduction
作物改良組作物改良組
中草藥研究組中草藥研究組
環保及工業用酵素環保及工業用酵素
動物疫苗技術動物疫苗技術
IntroductionIntroduction
Lymphocyte stem cell
( Bone marrow )
B cell ( Bone marrow )
T cell ( Thymus )
Tc
( Cytotxic T cell )
Th
( Helper T cell )
IntroductionIntroduction
Th1 cells activate macrophages. Th1 cells activate macrophages.
Enabling them to destroy intracellular Enabling them to destroy intracellular
microorganisms more efficiently.microorganisms more efficiently.
Th2 cells drive B cells to differentiate Th2 cells drive B cells to differentiate
and produce immunoglobulins of all and produce immunoglobulins of all
other type. other type.
IntroductionIntroduction
TT 細胞由未受刺激的細胞由未受刺激的 Th0Th0 細胞發展到 細胞發展到 Th1Th1 細胞的細胞的
分化過程中,其分化過程中,其 Interferon gamma promoter (pIFInterferon gamma promoter (pIF
N-γ) N-γ) 會被活化而導致會被活化而導致 IFN-γIFN-γ 的產生。 的產生。
T-bet T-bet 可以促進 可以促進 TT 細胞從 細胞從 Th0 Th0 細胞分化到 細胞分化到 Th1 Th1
細胞,也可以控制 細胞,也可以控制 IFN-γ IFN-γ 的表現。 的表現。
IntroductionIntroduction
Th0 Th1 IFN-γT-bet
MaterialMaterialYeastYeast
Main difference between yeast and E.coli culMain difference between yeast and E.coli cultureture
YeastYeast E.ColiE.Coli
4~6 days4~6 days 16 hours16 hours
YeastYeast E.ColiE.Coli
3030℃℃ 3737℃℃
YeastYeast E.ColiE.Coli
YPDYPD LBLB
Incubation timeIncubation time
Incubation temperatureIncubation temperature
Culture mediumCulture medium
MethodMethodYeast One Hybrid systemYeast One Hybrid system
One-Hybrid System provides an in vivo genetic assay One-Hybrid System provides an in vivo genetic assay
used for isolating novel genes encoding proteins that used for isolating novel genes encoding proteins that
bind to a target, cis-acting regulatory elementbind to a target, cis-acting regulatory element
Protein-DNA interactionProtein-DNA interaction
由於 由於 T-bet T-bet 可和 可和 IFN-IFN-γγ 啟動子結合並且調控之,啟動子結合並且調控之,
因此想利用 因此想利用 T-bet T-bet 和 和 IFN-γIFN-γ 啟動子的作用來探討 啟動子的作用來探討
Yeast one hybrid Yeast one hybrid 系統是否可以偵測到兩者的作系統是否可以偵測到兩者的作
用。若可行,我們將利用此系統進一步來篩選其用。若可行,我們將利用此系統進一步來篩選其
他能和 他能和 IFN-IFN-γγ 啟動子作用的基因,與研究這些基啟動子作用的基因,與研究這些基
因的產物是否可以促進 因的產物是否可以促進 T cell T cell 從 從 Th0 Th0 細胞分化細胞分化
到 到 Th1 Th1 細胞。細胞。
AD
AD
BD
AD
BD
報導基因BD
AD BDAD BD
ADAD
AD
BDAD
BD
Reporter geneBD
c. Activation
b. Fusion protein binding
a. Fusion protein expression
AD BDAD BDAD BDAD BD
Yeast chromosome
Constructed plasmid
T-bet
IFN-r promoter
GAL4AD
XbaI
IFN-r promoter
EcoRI
pHISi6786 bp
HIS3
URA3
AmpR
Bam HI (1447)
Eco RI (2)
Nco I (2935)
Sma I (9)
Xba I (43)
Xho I (1009)
Xma I (7)
Bst XI (542)
Eco RV (2918)
Kpn I (775)
Sac I (17)
Hin dIII (457)
Hin dIII (644)
Hin dIII (3635)
Pst I (836)
Pst I (2712)
Pst I (3627)
ligation
Reporter gene
T-bet binding domain
EcoRI PstIpGAD4246659 bp
LEU2
AmpR
GAL4AD
Bam HI (844)
Eco RI (834)
Pst I (860)
Sma I (841)
Xma I (839)
Eco RV (1891)
Kpn I (484)
Hin dIII (410)
Hin dIII (1105)
Bst XI (767)
Bst XI (1130)
Bst XI (1930)
Activation domain
ligation
Report gene
Yeast strain : YM4271 ( MATa )、 Y187 ( MATα )
Can’t synthesis histidine 、 leucine 、 uracil by itself 。
Yeast
Incubate
Yeast selection medium
-His or -Leu or -Ura
Yeast can’t growth on selection medium
pHISi-IFN-γ
Yeast ( YM4271 )
Yeast selection medium
-His
pGAD424-T-bet
Yeast ( Y187 )
Yeast selection medium
-Leu
Yeast selection medium
-His
Appropriate 3-AT
3-AT : a inhibitor of the HIS3 gene product
Yeast ( YM4271 )
pHISi-IFN-γ
Yeast selection medium
-His/-Leu
Yeast ( Y187 )
pGAD424-T-bet
Yeast can’t growth
Yeast ( YM4271 )
pHISi-IFN-γ
Yeast can’t growth
Yeast selection medium
-His/-Leu
Incubate
Incubate
pHISi-IFN-γ
pGAD424
Appropriate 3-AT
Yeast selection medium
-His/-Leu
Yeast mating
YM4271 Y187
Yeast selection medium
-His/-Leu
pHISi-IFN-γ
pGAD424-T-bet
Y187
YM4271
Future work
Screen library to find new transcription Screen library to find new transcription factor.factor.
THE ENDThank you