8 酵母双杂交等

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LEU 2+
GAL4 Promoter
Reporter
LACZ
Fig. 12-35 The yeast two-hybrid system for detecting gene interaction. The system uses the binding of two protein under test to restore the function of the GAL4 protein , which activates a reporter gene.
2μori CamR GAL4 binding dom CamR BD “ Bait ” protein 2μori GAL4 activation domain AD “ Target ” protein
TPR 1+ Unite Interaction Target GAL4 AD Bait GAL4 BD
酵母双杂交系统
Yeast Two-hybrid System(interaction trap) A System to Identify Proteins Involved in Specific Protein-protein Interactions
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
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。
Yeast two hybrid vectors
酵母单杂交的原理与应用实例 OneThe Mechanism and Application of Yeast Onehybrid 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.
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

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。
AD His Bait elements lacZ Reporter(s)
Fra Baidu bibliotek
•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
The proximal NPT2 promoter sequences and the location of phosphate responsive element (PRE).
Cloning of DNA-binding proteins using the yeast one-hybrid system. A reporter gene, NPT2 PRE-CYC1HIS3, for yeast one-hybrid study was constructed as follows: The S. cerevisiae HIS3 coding region connected downstream of the UASless S. cerevisiae CYC1 promoter was constructed on a pUC19 basedplasmid containing the S. cerevisiae ADE2 gene fragment. This plasmid was designed as pCHNaPi0. The five tandem copies of 36 bp doublestranded oligonucleotide, which originates from the sequences corresponding to the nucleotide positions from -1010 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.
激活区 DNA 结合区 转录因子
5’ 调控位点
启动子
结构基因
待测蛋白 ?
报告基因
Activation Domain Prey Protein Bait Protein Binding Domain
Reporter Gene
Yeast one-hybrid system
Protein-DNA interaction
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