Patrick F.H. Lai the Graduate Student
pfhlai at LOOKSMART.COM
Tue Sep 22 03:57:55 EST 1998
---- Begin Original Message ----
I have cloned by 3'RACE-PCR the human homologue of a rat protein
that is supposed to be isoprenylated / carboxymethylated based on
the CVVL amino acid consensus sequence at the very C-terminus.
Though the newly cloned human homologue is quite different at the
C-terminal side it also has a similar consensus (CVLL). What I do not
understand is that actually the part of the untranslated 3'-region is
somewhat conserved between rat and human. Is that common?
Could that have to do with the isoprenylation e.g. could there be
sequences in the 3' region that influence isoprenylation?
Normally I would think that this is just a matter of the protein,
With DNA-blast searches I pick up the rat homologue with the
first half of the coding sequence. The second half of the coding
sequence is too different for blast to pick up the rat homologue and
surprisingly this is possible with only the 3' untranslated
DNA sequence from the Stop codon to the poly A tail.
I plan to express the cDNA in COS and Sf9 cells and because of
this sequence conservation I am hesitant to just leave the 3' UTR
out in the constructs (as I would normally do.)
I am grateful for any advice or personal experiences with the
conservation of 3' untranslated regions of 'your' sequences and
any opinion on whether the sequence after the Stop codon
matters for heterologous expression.
Thanks a lot for your help
---- End Original Message ----
Yes, the 3'UTR is very commonly conserved.
This region is believed to be involved in the regulation of transcript
stability (=> steady state level of expression) and intracellular
transport / localization of the transcript, as well as polyadenylation
In some cases, it enhances translation, too.
Why do you want to express your gene of interest ?
Just to see what the gene product does ?
Then I don't think adding the 3'UTR helps.
Check the sequence. If it has many copies of AUUUA etc., don't
include them -- they often code for rapid degradation of mRNA.
(if you don't believe me, search for 'AUUUA' in medline.)
Common expression vectors usually give the 'transgene' a 3'UTR from
another gene to enhance expression, e.g. part of the bovine growth
hormone 3'UTR... and label that as the polyadenylation signal.
However, if you are generating transgenic animals, it's a completely
(I am trying to convince my boss to let me work on this......
but I don't think he will...... )
Hope this is useful info. :-)
Patrick F.H. Lai < PFHLai at looksmart.com >
University of Toronto
Toronto, Ontario, Canada
or keep looking.
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