W*n
2 楼
想获取一个一个物种的某基因A的序列，从其5'UTR及开始的25个编码区碱基设计
Morpholino序列用于基因阻断。 从朋友那边（正在做此物种测序）获取了一包含这个
基因的测序序列：包含这个基因的所有内含子，外显子以及启动子区域 （只是中间也
有几个大片的NNNNN区）。我通过将其和另外一个亲缘最近的同属的其他物种（已经测
序）进行比较，找出了编码区的内含子和外显子（因为蛋白序列高度相似）。
现在的问题是：怎样从原始序列中找出5'UTR的确切序列？ 这个5'UTR是否一定在第一
个编码区的外显子上，还是可能在这个外显子的上游还可能有一个外显子，用来剪贴出
全部或者部分5'UTR？ 怎样判断5'UTR的起始位点？
请问用什么软件可以分析？谢谢！
Morpholino序列用于基因阻断。 从朋友那边（正在做此物种测序）获取了一包含这个
基因的测序序列：包含这个基因的所有内含子，外显子以及启动子区域 （只是中间也
有几个大片的NNNNN区）。我通过将其和另外一个亲缘最近的同属的其他物种（已经测
序）进行比较，找出了编码区的内含子和外显子（因为蛋白序列高度相似）。
现在的问题是：怎样从原始序列中找出5'UTR的确切序列？ 这个5'UTR是否一定在第一
个编码区的外显子上，还是可能在这个外显子的上游还可能有一个外显子，用来剪贴出
全部或者部分5'UTR？ 怎样判断5'UTR的起始位点？
请问用什么软件可以分析？谢谢！
m*5
3 楼
RLM 5'RACE
z*6
4 楼
我还一直以为mRNA数据库里面CDS前面的就是5‘UTR呢。。。惭愧。。。
W*n
5 楼
mRNA数据库里面CDS前面的那部分肯定就是5‘UTR， 因为我觉得那些mRNA都是剪切过的
成熟mRNA.现在我所序列是从基因组测序出来的原始序列，我需要首先确定这个基因表
达的时候，从DNA转录到RNA从哪个位点开始的，然后转录出的RNA在哪些地方剪切去掉
内含子，然后剩下的就是成熟mRNA.
【在 z*******6 的大作中提到】
: 我还一直以为mRNA数据库里面CDS前面的就是5‘UTR呢。。。惭愧。。。
成熟mRNA.现在我所序列是从基因组测序出来的原始序列，我需要首先确定这个基因表
达的时候，从DNA转录到RNA从哪个位点开始的，然后转录出的RNA在哪些地方剪切去掉
内含子，然后剩下的就是成熟mRNA.
【在 z*******6 的大作中提到】
: 我还一直以为mRNA数据库里面CDS前面的就是5‘UTR呢。。。惭愧。。。
W*n
6 楼
这是个好办法，但是我觉得通过基因组测序的序列分析，也应该大致找出来需要的序列。
【在 m******5 的大作中提到】
: RLM 5'RACE
【在 m******5 的大作中提到】
: RLM 5'RACE
s*s
7 楼
don't think so
列。
【在 W*********n 的大作中提到】
: 这是个好办法，但是我觉得通过基因组测序的序列分析，也应该大致找出来需要的序列。
列。
【在 W*********n 的大作中提到】
: 这是个好办法，但是我觉得通过基因组测序的序列分析，也应该大致找出来需要的序列。
K*4
8 楼
I remember that some Japan groups have sequenced all UTRs of the genes and
transcript isoforms that generated by alternative splicing. They published a
lot of Nature and NG papers from this.
Dont know if they have made it public.
Go back to your purpose, I think you need to find TSS or splicing sites for
morpholino, not 5UTR, right?
【在 W*********n 的大作中提到】
: 想获取一个一个物种的某基因A的序列，从其5'UTR及开始的25个编码区碱基设计
: Morpholino序列用于基因阻断。 从朋友那边（正在做此物种测序）获取了一包含这个
: 基因的测序序列：包含这个基因的所有内含子，外显子以及启动子区域 （只是中间也
: 有几个大片的NNNNN区）。我通过将其和另外一个亲缘最近的同属的其他物种（已经测
: 序）进行比较，找出了编码区的内含子和外显子（因为蛋白序列高度相似）。
: 现在的问题是：怎样从原始序列中找出5'UTR的确切序列？ 这个5'UTR是否一定在第一
: 个编码区的外显子上，还是可能在这个外显子的上游还可能有一个外显子，用来剪贴出
: 全部或者部分5'UTR？ 怎样判断5'UTR的起始位点？
: 请问用什么软件可以分析？谢谢！
transcript isoforms that generated by alternative splicing. They published a
lot of Nature and NG papers from this.
Dont know if they have made it public.
Go back to your purpose, I think you need to find TSS or splicing sites for
morpholino, not 5UTR, right?
【在 W*********n 的大作中提到】
: 想获取一个一个物种的某基因A的序列，从其5'UTR及开始的25个编码区碱基设计
: Morpholino序列用于基因阻断。 从朋友那边（正在做此物种测序）获取了一包含这个
: 基因的测序序列：包含这个基因的所有内含子，外显子以及启动子区域 （只是中间也
: 有几个大片的NNNNN区）。我通过将其和另外一个亲缘最近的同属的其他物种（已经测
: 序）进行比较，找出了编码区的内含子和外显子（因为蛋白序列高度相似）。
: 现在的问题是：怎样从原始序列中找出5'UTR的确切序列？ 这个5'UTR是否一定在第一
: 个编码区的外显子上，还是可能在这个外显子的上游还可能有一个外显子，用来剪贴出
: 全部或者部分5'UTR？ 怎样判断5'UTR的起始位点？
: 请问用什么软件可以分析？谢谢！
W*n
9 楼
Thanks for the useful information.
For I want to use morpholino to block the traslation of the gene, not for
the splicing block, I think I know the 5'UTR and the starting 25bp coding
sequence--it is also required by the Morpholino company.
Thanks
a
for
【在 K**4 的大作中提到】
: I remember that some Japan groups have sequenced all UTRs of the genes and
: transcript isoforms that generated by alternative splicing. They published a
: lot of Nature and NG papers from this.
: Dont know if they have made it public.
: Go back to your purpose, I think you need to find TSS or splicing sites for
: morpholino, not 5UTR, right?
For I want to use morpholino to block the traslation of the gene, not for
the splicing block, I think I know the 5'UTR and the starting 25bp coding
sequence--it is also required by the Morpholino company.
Thanks
a
for
【在 K**4 的大作中提到】
: I remember that some Japan groups have sequenced all UTRs of the genes and
: transcript isoforms that generated by alternative splicing. They published a
: lot of Nature and NG papers from this.
: Dont know if they have made it public.
: Go back to your purpose, I think you need to find TSS or splicing sites for
: morpholino, not 5UTR, right?
m*5
10 楼
生物信息方法似乎不可信，找putative transcription start site还是5'RACE最可靠
列。
【在 W*********n 的大作中提到】
: 这是个好办法，但是我觉得通过基因组测序的序列分析，也应该大致找出来需要的序列。
列。
【在 W*********n 的大作中提到】
: 这是个好办法，但是我觉得通过基因组测序的序列分析，也应该大致找出来需要的序列。
n*7
11 楼
you mean FANTOM?
a
for
【在 K**4 的大作中提到】
: I remember that some Japan groups have sequenced all UTRs of the genes and
: transcript isoforms that generated by alternative splicing. They published a
: lot of Nature and NG papers from this.
: Dont know if they have made it public.
: Go back to your purpose, I think you need to find TSS or splicing sites for
: morpholino, not 5UTR, right?
a
for
【在 K**4 的大作中提到】
: I remember that some Japan groups have sequenced all UTRs of the genes and
: transcript isoforms that generated by alternative splicing. They published a
: lot of Nature and NG papers from this.
: Dont know if they have made it public.
: Go back to your purpose, I think you need to find TSS or splicing sites for
: morpholino, not 5UTR, right?
l*1
12 楼
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
----
【在 s******s 的大作中提到】
: don't think so
:
: 列。
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
----
【在 s******s 的大作中提到】
: don't think so
:
: 列。
K*4
13 楼
If you want to block the translation of the gene, then what you need is find
the Translation Start Site or region around CDS starting point?
Correct me if I am wrong.
【在 W*********n 的大作中提到】
: Thanks for the useful information.
: For I want to use morpholino to block the traslation of the gene, not for
: the splicing block, I think I know the 5'UTR and the starting 25bp coding
: sequence--it is also required by the Morpholino company.
: Thanks
:
:
: a
: for
the Translation Start Site or region around CDS starting point?
Correct me if I am wrong.
【在 W*********n 的大作中提到】
: Thanks for the useful information.
: For I want to use morpholino to block the traslation of the gene, not for
: the splicing block, I think I know the 5'UTR and the starting 25bp coding
: sequence--it is also required by the Morpholino company.
: Thanks
:
:
: a
: for
W*n
14 楼
sorry for my misunderstanding.
Yes, you are right, I need to find the Translation Start Site or region
around CDS starting point.
Now, I have found the Tranlation Start Site, and also I can make sure the
downstream coding sequences after the TSS. So now I need a comfirmed 5'UTR
sequence, at least some of the sequence near the TSS. Do you think the
unstream of the TSS in the gemome is 5'UTR ? or at least we can make sure of
a samll seuquence before the TSS?
I seed the Coding sequence and 300bp upstream sequence to a company to let
them help designing a Morpholino,and they chose a sequence from -6 to -30 (
6th base to 30 base upstream the TSS in the genome). I am not sure if it is
a correct 5"UTR. So can you tell me if I can make sure the sequence is
really part of the 5'UTR?
The sequence is as following: the sequence in[ ]is for Morpholino, (ATG) is
the TSS,
TTTGGCCTAAGGCGGGGCA[AAAGCACAGCAAAGCAAACGACCAA]CCATA(ATG)
GCCAACTTTTACCGTACCGAGA
the sequence of the upstream and downstream of the TSS are:(lower case for
upstream of TSS, upper case for TSS and coding sequence)
cggtgcgtgggacaaagcggttgtcccccggttgttgagataaataagaataggtgtcaattctgtgtgtgtgtgt
gggtatttgtgcttatgctgaaagtgtgcgtgcctgtgtgtgtccatctgttccgtaataaatagaaatatcagct
agagaaaacaacacccgcaagcggcggagacaaaatttggcctaaggcggggcaaaagcacagcaaagcaaacgac
caaccataATGGCCAACTTTTACCGTACCGAGATCAACAAAACGGAATGGGAAGTTCCGGACAAGTATCaAGCACT
GAcCCCGGTCGGTAGCGGTGcCTACGGGCAGGTGTG
So do you think we can use designed part for the gene's blocking ? Thanks!
find
【在 K**4 的大作中提到】
: If you want to block the translation of the gene, then what you need is find
: the Translation Start Site or region around CDS starting point?
: Correct me if I am wrong.
Yes, you are right, I need to find the Translation Start Site or region
around CDS starting point.
Now, I have found the Tranlation Start Site, and also I can make sure the
downstream coding sequences after the TSS. So now I need a comfirmed 5'UTR
sequence, at least some of the sequence near the TSS. Do you think the
unstream of the TSS in the gemome is 5'UTR ? or at least we can make sure of
a samll seuquence before the TSS?
I seed the Coding sequence and 300bp upstream sequence to a company to let
them help designing a Morpholino,and they chose a sequence from -6 to -30 (
6th base to 30 base upstream the TSS in the genome). I am not sure if it is
a correct 5"UTR. So can you tell me if I can make sure the sequence is
really part of the 5'UTR?
The sequence is as following: the sequence in[ ]is for Morpholino, (ATG) is
the TSS,
TTTGGCCTAAGGCGGGGCA[AAAGCACAGCAAAGCAAACGACCAA]CCATA(ATG)
GCCAACTTTTACCGTACCGAGA
the sequence of the upstream and downstream of the TSS are:(lower case for
upstream of TSS, upper case for TSS and coding sequence)
cggtgcgtgggacaaagcggttgtcccccggttgttgagataaataagaataggtgtcaattctgtgtgtgtgtgt
gggtatttgtgcttatgctgaaagtgtgcgtgcctgtgtgtgtccatctgttccgtaataaatagaaatatcagct
agagaaaacaacacccgcaagcggcggagacaaaatttggcctaaggcggggcaaaagcacagcaaagcaaacgac
caaccataATGGCCAACTTTTACCGTACCGAGATCAACAAAACGGAATGGGAAGTTCCGGACAAGTATCaAGCACT
GAcCCCGGTCGGTAGCGGTGcCTACGGGCAGGTGTG
So do you think we can use designed part for the gene's blocking ? Thanks!
find
【在 K**4 的大作中提到】
: If you want to block the translation of the gene, then what you need is find
: the Translation Start Site or region around CDS starting point?
: Correct me if I am wrong.
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