I am trying to express a plasmodium protein in yeast whose gene is 2 kb but when I do RT PCR I get a 450 bp band. This was sequenced and I found that it has initial 430 bp followed by final 20 bp of my gene. All introns have already been removed during cloning itself. Is the transcript getting aberrantly spliced? How is this possible? I also don't see expression of my protein due to this.
does the sequence you are missing starts with GT and ends in AG? In that case it is treated like an intron. You can do some mutagenesis to replace the GT/AG dinucleotide while keeping the codon synonymous
The missing sequence starts with TT and ends with CA...
I think they are non canonical splice sites.
Have a look at this paper:
Nucleic Acids Res. 2001 Jun 15;29(12):2581-93. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions. Thanaraj TA1, Clark F.
you can still mutate them, but remember to keep the coding sequence unchanged by creating synonymous codons. Also be careful not to create new spice sites. If feasible, you can also change the nucleotides around TT and CA in order to destroy the consensus splice sites sequence.
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