Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Gene expression change after mutagenesis-- please help! - (Jul/12/2011 )

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!

-Gangwolf-

Gangwolf on Wed Jul 13 05:16:05 2011 said:


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.

-qzlabs-

EDIT: see below

-Gangwolf-

qzlabs on Thu Jul 14 04:59:12 2011 said:


Gangwolf on Wed Jul 13 05:16:05 2011 said:


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?

-Gangwolf-

Gangwolf on Mon Jul 18 09:34:28 2011 said:


qzlabs on Thu Jul 14 04:59:12 2011 said:


Gangwolf on Wed Jul 13 05:16:05 2011 said:


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?

-Gangwolf-