酵母双杂交系统 Yeast Two-hybrid System(interaction trap)

Presentation on theme: "酵母双杂交系统 Yeast Two-hybrid System(interaction trap)"— Presentation transcript:

1 酵母双杂交系统 Yeast Two-hybrid System(interaction trap)
A System to Identify Proteins Involved in Specific Protein-protein Interactions

2 Protein-protein interactions
Protein-protein interactions are intrinsic to virtually every cellular process ranging from DNA replication, transcription, splicing and translation, to secretion, cell cycle control, intermediary metabolism, formation of cellular macrostructures and enzymatic complexes. The formation of large cellular structures such as the cytoskeleton, the nuclear scaffold, and the mitotic spindle result from complex interactions between proteins. Relatively smaller structures such as nuclear pores, centrosomes and kinetochores are beginning to be characterized and, in each case, protein-protein interactions seem to play a crucial role.

3 各种转录调控因子氨基酸残基序列不同，但许多都含有两大结构区域，即DNA结合功能域和转录激活功能域，两者之间由一伸展性良好的长臂连接。DNA结合功能域识别相应的顺式元件序列并与之结合后，转录激活功能域借助长臂的伸缩，作用于转录基本因子，进而激活转录启动。

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9 Reporter mRNA Reporter mRNA Reporter mRNA Reporter mRNA Reporter mRNA
Activation Domain Prey Protein Reporter mRNA Bait Protein Reporter mRNA Reporter mRNA Binding Domain Reporter mRNA Reporter mRNA Reporter Gene

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13 酵母单杂交系统Yeast one-hybrid system
Protein-DNA interaction

14 clone element into a reporter construct and make stable yeast strain
transfect aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator if the fusion protein binds to your element then the reporter gene will be activated

15 酵母单杂交的原理与应用实例 The Mechanism and Application of Yeast One-hybrid System <<生物工程进展 >>2001年04期 陈峰 , 李洁 , 张贵友 , 刘强 许多诱导型基因的表达,都受特定的转录因子和顺式元件调控.要阐明各种信号传递途径与基因表达调控的机理,克隆和鉴定转录因子是关键.近年来酵母单杂交方法被广泛应用于克隆和鉴定各种动植物的转录因子,本文以拟南芥DREB转录因子的克隆为例,介绍酵母单杂交方法的原理和具体应用.

16 Transcriptional regulation of the NPT2 gene by dietary phosphate
Kidney International (2001) 60, 412–415; Ken-Ichi Miyamoto and Mikiko Itho Transcriptional regulation of the NPT2 gene by dietary phosphate. Dietary phosphate (Pi) is an important regulator for renal Pi reabsorption. The type II sodium-dependent phosphate (Na/Pi) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal Pi reabsorption. The yeast one-hybrid system was used to clone a transcription factor that binds to a specific sequence (Pi response element) in the promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor E3 (TFE3) were isolated. TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary Pi. Abbreviations: bp, base pair; EMSA, electrophoretic mobility shift assay; kb, kilobase pair; Na/Pi, sodium-dependent phosphate; NPT2, sodium-dependent phosphate cotransporter; OK, opossum kidney; Pi, inorganic phosphate; PRE, phosphate response element; PTH, parathyroid hormone; TFE3, mouse transcription factor E3; TGF- , transforming growth factor- ; UAS, upstream activating sequence

17 The proximal NPT2 promoter sequences and the location of phosphate responsive element (PRE).

18 Cloning of DNA-binding proteins using the yeast one-hybrid system
Cloning of DNA-binding proteins using the yeast one-hybrid system. A reporter gene, NPT2 PRE-CYC1-HIS3, for yeast one-hybrid study was constructed as follows: The S. cerevisiae HIS3 coding region connected downstream of the UAS-less S. cerevisiae CYC1 promoter was constructed on a pUC19 based-plasmid containing the S. cerevisiae ADE2 gene fragment. This plasmid was designed as pCHNaPi0. The five tandem copies of 36 bp double-stranded oligonucleotide, which originates from the sequences corresponding to the nucleotide positions from to -985 in human NPT2 promoter, including the Pi response element (PRE), were inserted into upstream of the CYC1 promoter on pCHNaPi0 as its UAS sequences8.

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20    在 one-hybrid assay 中(图中B）， AD直接融合至 DBD。 這個 assay 可用來尋找結合特殊DNA序列的DBDs。

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22 补充：其实我个人认为two-hybrid 或one-hybrid system, 翻译成双杂合或单杂合系统为宜。
安徽大学生命科学学院查向东整理