Question: Insertion in intron
0
gravatar for dadhokane
2.5 years ago by
dadhokane0
dadhokane0 wrote:

How can I explain an insertion of 158 bp in intronic region suppress the gene expression??

I have one resistant and one susceptible line, I sequenced a gene in both and found 158bp insertion in intronic region in the susceptible line, where the gene expression is significantly lower compared to resistant line. How can I explain this?

For note: I have sequenced 1kb promoter region upstream of ATG but wouldn't see any sequence varaition

Thanking you!

sequence • 847 views
ADD COMMENTlink modified 22 months ago by Biostar ♦♦ 20 • written 2.5 years ago by dadhokane0
2
gravatar for harold.smith.tarheel
2.5 years ago by
United States
harold.smith.tarheel4.3k wrote:

It's entirely possible to reduce/abrogate splicing or change the splice acceptor site (e.g., by altering the polypyrimidine tract or lariat branch site), which typically results in out-of-frame transcripts that are subject to nonsense-mediated decay (NMD).

Point of clarification: by CDS sequencing, do you mean genomic DNA or RNA-derived cDNA? The former cannot not detect splicing errors or NMD, and even RNA-Seq may not be sufficiently sensitive for aberrant splicing (and NMD manifests as reduced expression).

ADD COMMENTlink modified 2.5 years ago • written 2.5 years ago by harold.smith.tarheel4.3k

I sequenced cDNA using Sanger sequencing

ADD REPLYlink written 2.5 years ago by dadhokane0

Since it's a heterozygous variant, could you see both alleles when sequencing the cDNA?

ADD REPLYlink written 2.5 years ago by WouterDeCoster38k

No but just I have one allele sequenced, I can see the difference on gel

ADD REPLYlink written 2.5 years ago by dadhokane0
2

???

I'm confused; are you saying that the cDNA is a different size by gel electrophoresis? Then how can the cDNA sequence be identical to the wild-type?

ADD REPLYlink modified 2.5 years ago • written 2.5 years ago by harold.smith.tarheel4.3k
1

I agree it's not clear, likely a terminology problem, that's why I more explicitly reformatted my question.

ADD REPLYlink modified 2.5 years ago • written 2.5 years ago by WouterDeCoster38k

So the allele carrying the insertion is not expressed?

ADD REPLYlink written 2.5 years ago by WouterDeCoster38k
1
gravatar for skbrimer
2.5 years ago by
skbrimer530
United States
skbrimer530 wrote:

I guess it could be a couple things. Like the RBS has moved and the reading frame is destroyed. But that would lead to a total knock out of the protein. It could be causing a splicing error can be giving you a truncated or mutated protein that is still being made but less effective. If you have any RNAseq data you could use that as well to find more evidence for this insert causing your mutation.

ADD COMMENTlink written 2.5 years ago by skbrimer530
1

Splicing would be my primary suspect.

@OP: is this insertion homozygous or heterozygous? Which organism?

ADD REPLYlink written 2.5 years ago by WouterDeCoster38k

But no difference when I sequenced CDS in both, its exactly the same.

Its in wheat, its heterozygous

ADD REPLYlink written 2.5 years ago by dadhokane0

Hello

I agree with your assumptions, but I have sequenced the CDS and would not see any difference within the CDS in both the lines. Some other reason like downstream regulatory elements or sRNAs suppressing the gene expression? Your suggestions?

Thanking you

ADD REPLYlink written 2.5 years ago by dadhokane0
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1525 users visited in the last hour