about splicing - who knows? (Feb/09/2007 )
Hi,
Does anyone know if the first exon is found in every splicing variant?
I have 2 bands in a NB...one of the protein I know and a bigger one that I imagine it could be a splicing alternative......so based on this info I want to get its sequence and caracterize it....but how should I start?
Could it be possible to recover mRNA, change it into cDNA and then do PCR with poly T and a part of exon 1 as primers?
The known sequence of the smaller band (some years ago we recover the sequence that codes for a protein induced during stress by doing a library) has 12 exons and I suppose that if the larger band in the NB is from a splicing variant it might have exons in common (in fact it has to, otherwise the probe wouldn't have hibridize with it)...... so how can I do to study the splicing based on this information?
If there is only one promoter, the transcription and translation start at the same place in each splicing variant? ( that would mean if that's the case, both species have the same first exon) do you know?
....And do you have any idea of the probability to have splicing due to different promoters?? I mean; what is more common: splicing due to different promoters? or because of other reasons different than the promoter region?
Any ideas?
Hi Biotech,
There is a reasonable chance that the additional mass you are finding is due to an intron inclusion. Does the mass increase correspond to the mass of any known introns?
Regards,
- Jon
not all variants have to have the first exon - but it could depend on the gene
try some 5' and 3' RACE to check this sort of thing out.
good luck
Does anyone know if the first exon is found in every splicing variant?
I have 2 bands in a NB...one of the protein I know and a bigger one that I imagine it could be a splicing alternative......so based on this info I want to get its sequence and caracterize it....but how should I start?
Could it be possible to recover mRNA, change it into cDNA and then do PCR with poly T and a part of exon 1 as primers?
The known sequence of the smaller band (some years ago we recover the sequence that codes for a protein induced during stress by doing a library) has 12 exons and I suppose that if the larger band in the NB is from a splicing variant it might have exons in common (in fact it has to, otherwise the probe wouldn't have hibridize with it)...... so how can I do to study the splicing based on this information?
If there is only one promoter, the transcription and translation start at the same place in each splicing variant? ( that would mean if that's the case, both species have the same first exon) do you know?
....And do you have any idea of the probability to have splicing due to different promoters?? I mean; what is more common: splicing due to different promoters? or because of other reasons different than the promoter region?
Any ideas?
I know of plenty of cases where the first exon is a splice variant. About the most extreme is the transcriptional co-repressor, CtBP2. Using the first exon, and without alternate splicing, the protein is about 48 kDa, expressed in lots of tissues, the protein is in the nucleus, and controls transcription. If you use the alternate start codon and alternate first exon, the protein is called RIBEYE, is about 100 kDa, is cytoplasmic, is only expressed in the retina, cochlea and pineal gland, and helps shunt synaptic vesicles at very high speed onto the synapse! The two "first" exons have two distinct promoters, so the debate is on about whether you are talking about one gene with two different 1st exons, or 2 genes, expressed in different tissues for different purposes, but with the same downstream sequences!