m******5
发帖数: 1383 | 1 原理上来说，是否RNA ligase mediated 5'RACE得到的一定是有效的5‘末端，也就是
individually transcripted，而非降解或者unspecific splicing产物？
我目前的理解是这样的，但因为结果太奇怪，上来问一下.
另外，还有一个问题,putative ATG前面允许存在Exon-Exon junction么？ | a********k
发帖数: 2273 | 2 不一定
有很多基因两篇paper里面的RACE结果都不一样的，当然不能排除alternative
splicing和降解的情况
【在 m******5 的大作中提到】
: 原理上来说，是否RNA ligase mediated 5'RACE得到的一定是有效的5‘末端，也就是
: individually transcripted，而非降解或者unspecific splicing产物？
: 我目前的理解是这样的，但因为结果太奇怪，上来问一下.
: 另外，还有一个问题,putative ATG前面允许存在Exon-Exon junction么？
| m******5
发帖数: 1383 | 3 Reference?
As far as l know, Alternative splice and degrading don't add cap right?
【在 a********k 的大作中提到】
: 不一定
: 有很多基因两篇paper里面的RACE结果都不一样的，当然不能排除alternative
: splicing和降解的情况
| a********k
发帖数: 2273 | 4 not if the alternative splice is the first exon
I cannot image a degrading RNA w/ different caps, though.
【在 m******5 的大作中提到】
: Reference?
: As far as l know, Alternative splice and degrading don't add cap right?
| m******5
发帖数: 1383 | 5 So I can exclude the possibility of degradation but insider seriously about
splicing?
On the other hand, it is quite not possible to have a EJC in front of the
atg right? Since 40s alone can not remove EJC
【在 a********k 的大作中提到】
: not if the alternative splice is the first exon
: I cannot image a degrading RNA w/ different caps, though.
| m******5
发帖数: 1383 | 6 人工置顶一下，因为得到的结果和以前文章发表的用非RLM mediated的race结果都不一
样 | l**********1
发帖数: 5204 | 7 RE LZ
just go to
htp://www.mitbbs.com/article_t/Biology/31627067.html
11f
一句话： 要确定某个表达蛋白(A物种) 的真正的5' UTR 范围碱基序列
最好将其包括启动子部分的碱基序列转接到一个非启动子功能的载体
然后转染到异种(B物种: 可以是A物种的近缘) 的Cell line 表达那个目标蛋白
再提取那个表达后的目标蛋白
如果后者同In vivo (A物种) 的生化物理性质完全一致,
那么其包括启动子部分的碱基序列 就是真正的5' UTR 范围碱基序列
[回复]
发信人: lotkaeuler11 (Fibonacci), 信区: Biology
标 题: Re: 急求：怎样从基因组序列中找出基因的5'UTR确切序列？
发信站: BBS 未名空间站 (Thu Jan 19 05:09:42 2012, 美东)
ZZ
老外的forum 有关的讨论帖 的最精彩的一句推论:
>So if I understood well, you suggest to lengthening the 5' end of the
construct including part of the promoter in a promoterless vector and then
transfect different cell lines and see in which one the entire transcript
will be expressed.
details pls go to
htp://molecularbiology.forums.biotechniques.com/viewtopic.php?f=2&t=31288
看看能否有助LZ的这问题的解决?
有一点务必牢记: without in vivo experimental data support for a new species
5'UTR information Blast
information is just a block of feces!
transcription starting site in plasmids
by eyes » Jul 06 2011 5:32 am
Hi guys,
I've cloned a gene with its 5'UTR within a pGL3-control vector in order to
evaluate the effect on an upstream ATG in protein translation regulation. In
order to be sure that all elements I was looking at were present in the
transfected cells, I tested the cDNA obtained after retrotranscription of
total RNA extracted by the cells with primer on the entire 5' UTR.
Unexpectedly of the about 600 bp of the 5'UTR at least half of them are not
transcribed. With the hypothesis that the vector itself could negatively
influence this result I make a similar construct within a pCDNA3.1 vector,
but the result did not change at all. Do you have any similar experience and
any suggestion? I need absolutely to get the entire 5'UTR. I think that one
idea could be to lengthen the 5'UTR beyond the TSS but I'm not sure this
will help.
Thanks in advance for your help
----
Re: transcription starting site in plasmids
by morkfromork » Jul 06 2011 1:41 pm
Hi relaxin,
A 5' UTR will contain parts of the core promoter (for some genes). Remember
that the initiator sequence is located at the -2 to +4 position and that
promoter elements such as the DPE are located at around +30. I agree it's
unlikely that a 5' UTR would direct accurate transcription initiation
because it lacks the upstream elements TATA, TFIIB-u/d. However, I think a
likely explanation of what is happening is that an alternative (or cryptic)
TSS in the UTR of the gene of interest is over-riding the CMV TSS
----
Re: transcription starting site in plasmids
by eyes » Jul 07 2011 9:38 am
Hi morkfromork,
thank you very much for your replay. The explanation you gave on my results
fit perfectly. I based the design of my experiments on literature data and I
make the same construct was described by another group using the pGL3-CV. I
've also used the same cell line so I was quiet sure that it would have been
easy to express the entire 5'UTR. More than one TSS have been described in
the literature but this one was the only one whose translation should be
influenced by a uORF.
So if I understood well, you suggest to lengthening the 5' end of the
construct including part of the promoter in a promoterless vector and then
transfect different cell lines and see in which one the entire transcript
will be expressed.
Thanks a lot. Your comments were very helpfully
Bye
----
roger
【在 m******5 的大作中提到】
: 人工置顶一下，因为得到的结果和以前文章发表的用非RLM mediated的race结果都不一
: 样
|
|
