发信人: clonist (dream on), 信区: Biology
标 题: Re: 基因表达一问
发信站: The unknown SPACE (Sun Mar 30 01:14:21 2003), 站内信件

question 1 pretty much answers itself. if the proteolytic frag
functions at the transcription level it may occupy its own
promoter binding region (most likely enhancer region) to prevent
transcription complex binding or it can function as dominant
negative to bind an essential, specific positive regulator for
its transcription. if it functions at the post transcription
level several differnt pathways may be involved such as
translationl inhibition, degradation of the protein, degradation
of the mRNA etc.
when the seq is available lots of exp can be done to distinguish
each hypothesis.
just give you some experiments to think about
1. overexpression both the proteolytic frag and the whole protein
in a commercial plasmid
2. northern blot or RPA to determine mRNA level. you may wanna
distinguish nuclear fraction and cyto fraction.
3. nuclear run-on to determine transcription initiation rate
4. western blot to determine the protein level

【 在 brights (忍冬) 的大作中提到: 】
: There is a mammalian protein whose expression is believed to be regulated in
: the following way: after the protein is translated, it is cleaved, and one of
: the proteolytic fragments is thought to negatively regulated the further
: expression of more protein.
: 1. Provide two hypotheses of how this regulation might work at the molecular
: level(i.e.,what might this proteolytic fragment be doing?)
: one hypothesis should address transcriptional regulation, and one hypothesis
: should address something that could be occurring at the post trancriptional
: level.
: 2.Assume that the gene for this protein has been cloned, and the lab has
: access to that clone.How to provide two experiments to address each
: hypothesis.


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