发信人: notch (大梦谁先觉，平生我不知), 信区: Biology
标 题: Re: Yeast two hybrid system?
发信站: The unknown SPACE (Wed Sep 13 14:32:55 2000), 站内信件

照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)

回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— ————————————
|A | binding domain | |B | activition domain |
--------------------- ------------------------
两个都是in-frame fusion，完全transcribed to protein. 这样如果AB bind each
other, binding domain and activition domain就被拉到一起，结合在DNA上，
activate这个transcriptional factor 所本应activate 的gene transcription.
一般我们就在这个transcriptional factor结合的地方下面加个reporter gene
这样如果reporter gene 表达，如lacZ就是菌落蓝色，就说明AB bind.如果不表达
AB就不bind.


【 在 notch (大梦谁先觉，平生我不知) 的大作中提到: 】
: //kick, why u take it for granted that u will use lacZ for reporter gene?
: hehe...
: I am not sure, but as I remember, your system is the 1-hybrid system
: 【 在 Ras (很颓的猪。) 的大作中提到: 】
: : example:
: : --------------------- ----------------------------
: : | bait | activator| + |fragment A | reporter gene |
: : --------------------- ----------------------------
: : Bait ait is the gene u r intestered in, and is attached to a
: : transcription activator. fragment A is one of those from a DNA library
: : which is attached to a reporter gene.
: : Basically you transfect both the bait construct and library construct
: : into yeast (a and alpha respectively) and let the mate (each yest should have
: : only one of those library DNA fragment). If the bait can bind with one of
: : the fragments, then the transcription activator can be brought to the reporter
: : gene and transcribe the reporter gene. The expression of the reporter gene
: : will lead to blue color of the specific yeast colony. Then u pick the blue
: : colony, grow up culture, and purify DNA from it. You got the DNA that can
: : interact with your bait.


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