13 replies to this topic

[paulcross]

Most papers I read  analysised "coding strand" methylation status. I am very confusing about that.

Since transcription always happened in “template strand”, so coding strand methylation has nothing to do with transcription.

Am I wrong?

I thought we should use template strand sequence to design the MSP/BSP primers instead of coding strand sequence.

[bob1]

The coding strand is the one with the gene sequence that then is transcribed to make the mRNA... I don't get your question.

[leelee]

bob1, on 02 September 2012 - 05:09 PM, said:

The coding strand is the one with the gene sequence that then is transcribed to make the mRNA... I don't get your question.

Its the non-coding strand that is used for the template for the mRNA, not the coding strand.

So I think what paulcross is asking is does (and how?) the methylation on the coding strand have any effect on the transcription of the template strand into mRNA. (is that what you mean, paul? )

[paulcross]

leelee, on 02 September 2012 - 10:45 PM, said:

bob1, on 02 September 2012 - 05:09 PM, said:

The coding strand is the one with the gene sequence that then is transcribed to make the mRNA... I don't get your question.

Its the non-coding strand that is used for the template for the mRNA, not the coding strand.

So I think what paulcross is asking is does (and how?) the methylation on the coding strand have any effect on the transcription of the template strand into mRNA. (is that what you mean, paul? )

YES！That‘s what I mean.
And I also want to ask some thing related to this: Why we search CpG islands in coding strand ? Coding strand never get transcribed. Why we always stick to it?

[Trof]

Actually if you imagine it on a double-helix CpG islands are on both strands

[paulcross]

Trof, on 04 September 2012 - 12:14 AM, said:

Actually if you imagine it on a double-helix CpG islands are on both strands

coding strand 5'----gcgcgcgcgcgc--........--cgcgcgcgcgcg---
template strand  5'----cgcgcgcgcgcg--........--gcgcgcgcgcgc---

One CpG island or TWO CpG islands?

[bob1]

Your example would be two, separated by an elipsis.  Ones on complementary strands would not be considered separate islands.

Doesn't methylation lead to histone modification and formation of chromatin... inhibiting separation and hence transcription.

[Trof]

@paulcross: I don't understand what you ask. CpG island should be at 200bp long with high percentage of GC and CpG sequence.  Your example is not. It can be within one island or not, still I don't see a point of counting them unless you want to compare between different organisms or whatever.

You asked why people search for them in coding strand, when it's not transcribed. Since CpG is a palindrome, it's always on both strands, so you can search whatever one, coding strands are mostly in the databases.

[paulcross]

Trof, on 04 September 2012 - 01:23 AM, said:

@paulcross: I don't understand what you ask. CpG island should be at 200bp long with high percentage of GC and CpG sequence.  Your example is not. It can be within one island or not, still I don't see a point of counting them unless you want to compare between different organisms or whatever.

You asked why people search for them in coding strand, when it's not transcribed. Since CpG is a palindrome, it's always on both strands, so you can search whatever one, coding strands are mostly in the databases.

Its just a schematic. I dont think I need to type 200 CG  twice!

What you said  "Since CpG is a palindrome, it's always on both strands"  is wrong.  CpG island in one strand, doesn't mean  it would appear in another strand.  One example is that  if coding strand are consisted of 200 CGs  ,   there would be 200 GCs in another strand. Since their  transcribe direction are the same( no need to explain ) ,  there would be no CpG island in template strand at all.

[paulcross]

bob1, on 04 September 2012 - 01:10 AM, said:

Your example would be two, separated by an elipsis.  Ones on complementary strands would not be considered separate islands.

Doesn't methylation lead to histone modification and formation of chromatin... inhibiting separation and hence transcription.

Methylation iinterrupted  the transcription factors binds to DNA , then lead to mRNA treanscription decrease.  That‘s why we focused on the TFBS methylation status in promoter region, in methylation study.

[Trof]

@paulcross: I'm sorry, but you should refresh your DNA basics.
If you have CG on one strand 5'->3', you have GC on the other strand, but 3'->5', that means CG 5'->3'. And that's the same on both strands.



Methylation is on both strands too, since it takes place in the cytosine of CpG site. After replication of DNA, the single stranded methylation marks are quickly recognised and methylated on the daughter strand, which leads to preservation of the methylation pattern through division.

[paulcross]

Trof, on 04 September 2012 - 04:48 AM, said:

@paulcross: I'm sorry, but you should refresh your DNA basics.
If you have CG on one strand 5'->3', you have GC on the other strand, but 3'->5', that means CG 5'->3'. And that's the same on both strands.



Methylation is on both strands too, since it takes place in the cytosine of CpG site. After replication of DNA, the single stranded methylation marks are quickly recognised and methylated on the daughter strand, which leads to preservation of the methylation pattern through division.

You got it all wrong！

I will explained it in a real  gene sequence.

Human Gene SUMO1 (uc002uza.1)  NM_001005782


The first several bases of  SUMO1  Exon1.

...coding strand ---------------tgcgcgaaGCGGAAGTGACGCGAGG
template strand ---------------acgcgcttCGCCTTCACTGCGCTCC  

You can see that coding strand sequence start from tgcgcg  to  GAGG(left to right) , like the mRNA sequence.  We use this direction and sequence to search  CpG island !  


Template strand are about to bind with transcription factors  to start the TRANSCRIBE! So  its direction also  start from  left  to right.  That is the direction gene transcription goes on.  

If   the template strand  start form right to left , SUMO1 gene wont get expressed.  It make no sense.

Edited by paulcross, 04 September 2012 - 05:37 AM.

[Trof]

This is your SUMO1 mRNA sequence (same as coding sequence).
If you search in page for your "GCGGAAGTG" from your first line, you will find it.

Now select Show reverse complement from the right Customize view menu. This should be your template strand. Try to find there your "CGCCTTCAC" from your second line. You will not. Because that's a 3'->5' direction what you have.
You will only find it's reverse orientation "CACTTCCGC" there. Because all sequences are read 5'->3'.

5' tgcgcgaaGCGGAAGTGACGCGAGG 3' coding
3' acgcgcttCGCCTTCACTGCGCTCC 5' template

That's the reality of your sequence. If the coding strand separates the polymerase will create new strand also in the 5'->3' direction, so it will actually move on the template strand in the 3'->5' direction. This is because polymerase can add nucleotides to existing 3' end only and the strands have to be anti-parallel.


5' tgcgc3' polymerase->   3' new strand
3' acgcgcttCGCCTTCACTGCGCTCC 5' template

I know it can be confusing, but you better check it out in some book before you make some serious mistakes in your work.

[paulcross]

Trof, on 04 September 2012 - 06:04 AM, said:

This is your SUMO1 mRNA sequence (same as coding sequence).
If you search in page for your "GCGGAAGTG" from your first line, you will find it.

Now select Show reverse complement from the right Customize view menu. This should be your template strand. Try to find there your "CGCCTTCAC" from your second line. You will not. Because that's a 3'->5' direction what you have.
You will only find it's reverse orientation "CACTTCCGC" there. Because all sequences are read 5'->3'.

5' tgcgcgaaGCGGAAGTGACGCGAGG 3' coding
3' acgcgcttCGCCTTCACTGCGCTCC 5' template

That's the reality of your sequence. If the coding strand separates the polymerase will create new strand also in the 5'->3' direction, so it will actually move on the template strand in the 3'->5' direction. This is because polymerase can add nucleotides to existing 3' end only and the strands have to be anti-parallel.


5' tgcgc3' polymerase->   3' new strand
3' acgcgcttCGCCTTCACTGCGCTCC 5' template

I know it can be confusing, but you better check it out in some book before you make some serious mistakes in your work.

I see ！

I paid too much attention on  the  actual  mRNA transcribing direction, but its irrelevant  to  DNA methylation.


THANK YOU!

Edited by paulcross, 04 September 2012 - 06:37 AM.