Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Gene expression change after mutagenesis-- please help!


  • Please log in to reply
4 replies to this topic

#1 Gangwolf

Gangwolf

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 41 posts
1
Neutral

Posted 12 July 2011 - 09:16 PM

Hi Everyone!
I am studying a transcription factor with multiple serine phosphorylation sites. The interesting thing is that when I mutate some of the sites from serine to alanine using site directed mutagenesis, some of the mutants when transiently overexpressed in HEK293T cells, show significantly reduced mRNA expression compared to wild type plasmid. What are the possible regulatory mechanisms behind this altered expression?
With my superficial background in transcription regulation, I had no clue that a single or double base pair substitution mutation could have this considerably effects. Thanks in advance!

#2 qzlabs

qzlabs

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 13 July 2011 - 08:59 PM

Hi Everyone!
I am studying a transcription factor with multiple serine phosphorylation sites. The interesting thing is that when I mutate some of the sites from serine to alanine using site directed mutagenesis, some of the mutants when transiently overexpressed in HEK293T cells, show significantly reduced mRNA expression compared to wild type plasmid. What are the possible regulatory mechanisms behind this altered expression?
With my superficial background in transcription regulation, I had no clue that a single or double base pair substitution mutation could have this considerably effects. Thanks in advance!

Normally, transcription is controlled by the promoter, CMV in most cases. Your mRNA levels should not be changed if you had only substitutions in the gene.
For higher yield and more protein, check out the new bioreactors at https://qizhonglabs.com/main/.

#3 Gangwolf

Gangwolf

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 41 posts
1
Neutral

Posted 18 July 2011 - 01:32 AM

EDIT: see below

Edited by Gangwolf, 18 July 2011 - 01:35 AM.


#4 Gangwolf

Gangwolf

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 41 posts
1
Neutral

Posted 18 July 2011 - 01:34 AM


Hi Everyone!
I am studying a transcription factor with multiple serine phosphorylation sites. The interesting thing is that when I mutate some of the sites from serine to alanine using site directed mutagenesis, some of the mutants when transiently overexpressed in HEK293T cells, show significantly reduced mRNA expression compared to wild type plasmid. What are the possible regulatory mechanisms behind this altered expression?
With my superficial background in transcription regulation, I had no clue that a single or double base pair substitution mutation could have this considerably effects. Thanks in advance!

Normally, transcription is controlled by the promoter, CMV in most cases. Your mRNA levels should not be changed if you had only substitutions in the gene.

Thanks for the answer!
Good point! So far I've only checked the gene region by sequencing, but then I'd better make sure that the CMV promoter region is not accidently affected by mutagenesis.
So, are there really no know transcription regulation mechanisms that can be triggered by a single or double point mutation in the gene sequence?

#5 Gangwolf

Gangwolf

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 41 posts
1
Neutral

Posted 19 July 2011 - 02:42 AM



Hi Everyone!
I am studying a transcription factor with multiple serine phosphorylation sites. The interesting thing is that when I mutate some of the sites from serine to alanine using site directed mutagenesis, some of the mutants when transiently overexpressed in HEK293T cells, show significantly reduced mRNA expression compared to wild type plasmid. What are the possible regulatory mechanisms behind this altered expression?
With my superficial background in transcription regulation, I had no clue that a single or double base pair substitution mutation could have this considerably effects. Thanks in advance!

Normally, transcription is controlled by the promoter, CMV in most cases. Your mRNA levels should not be changed if you had only substitutions in the gene.

Thanks for the answer!
Good point! So far I've only checked the gene region by sequencing, but then I'd better make sure that the CMV promoter region is not accidently affected by mutagenesis.
So, are there really no know transcription regulation mechanisms that can be triggered by a single or double point mutation in the gene sequence?


Sequencing results show that the CMV promoter region does not contain any random mutations.
Is it possible that this transcription factor is regulating itself? Could that occur in a transient transfection cell model?




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.