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 role.
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。
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
酵母单杂交系统Yeast
one-hybrid system
Protein-DNA interaction
AD
His
Bait elements
lacZ
Reporter(s)
•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
酵母单杂交的原理与应用实例
The 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
•
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.
•
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。
• 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