[BiO BB] from kiran kumar

[Toshinori Endo]

Hi kiran,

Here is the abstract of the method todetermine the exon 1,
which you can find the detailed description in a standard
textbook like molecular biology of the gene or a cook book
like molecular cloning.

There are severals ways to confirm the exon 1.
First is to compare the sequence with the 5' end of cDNA
or mRNA by in silico comparison or by hybridization.

Second is to determine whether or not it is the true end,
S1 mapping may be performed, which is based on the hybridization
between genomic DNA and messenger RNA followed by the 
digestion of single stranded RNA by S1 RNase.  This method
can be applied to determine splice sites as well as 3' end.
Another method may also be employed, that is primer extension.

The third is to perform in vitro transcription to compare
the size of spliced form of the transcript with that of mRNA
by electrophoresis which ensures the there are no missing
fragment in the genomic clone.

They may sometime perfom in vitro tranlation as well.
If they have isolated protein sample which is relatively
rare case in the recent study, they compare the
molecular weight of translated product with native protein.
Otherwise, they may obtain the antibody somehow,
so that they can compare the MW of experimentally obtained 
protein with that of native one.

Those information will not appear in GenBank annotation.
You can find them only in literatures.  In addition, not many
researchers cares whether or not they have true 5' end
as long as the have sequence contain whole coding
sequence, which may be comfirmed by in vitro translation
described above.

Note that exon 1 does not necessary code the initiation codon.
That is, it may contain just a 5'UTR of the gene.  In such a case,
coding sequence starts in the second exon or even in the third.

Toshinori Endo


> -----Original Message-----
> From: bio_bulletin_board-admin at bioinformatics.org 
> [mailto:bio_bulletin_board-admin at bioinformatics.org]On Behalf Of 
> kiran challapalli
> Sent: Monday, November 19, 2001 6:51 PM
> To: bio_bulletin_board at bioinformatics.org
> Subject: [BiO BB] from kiran kumar
> 
> 
> Hello everyone,
> I am working with eukaryotic promoter analysis. could
> anyone explain me how exon1 of eukaryotic genes could
> be characterised or annotated by wet lab experiments.
> I mean how experimentally exon1 can be proved of a
> given sequence.
> Thank you in advance.
> Kiran kumar
> 
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