[Zbrafish] Re: Sequence homology for protein expression - help!

[dykvar from yahoo.com]

On Apr 20, 8:07 am, Joanna Wilson <joanna.wil... from mcmaster.ca> wrote:
> Hi there,
> I am totally unsurprised that you are finding sequence differences  
> with the gene you cloned and what is in NCBI.  We see this in the  
> genes we clone in the lab - one to 10 nucleotide sequence differences  
> (our genes are 1500 bp) with aa changes.  You can see this if you pull  
> out sequence information from NCBI for which there are multiple  
> entries - they will not be completely identical.  Some of this is  
> strain differences.  It doesn't mean it won't be expressed.  You need  
> to be sure that the differences are real (sequence several clones so  
> that you can show it isn't a PCR or sequencing error) and that it will  
> translate in frame without stops.
> Joanna Wilson
>
> Re:
> Message: 1
> Date: Fri, 18 Apr 2008 11:34:55 -0700 (PDT)
> From: dyk... from yahoo.com
> Subject: [Zbrafish] Sequence homology for protein expression - help!
> To: bionet-organisms-zebraf... from net.bio.net
> Message-ID:
>         <9da02037-1cd7-4aee-a587-463a24627... from e39g2000hsf.googlegroups.com>
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>
> Hi,
> I have a general question regarding rescue and protein expression via
> construct in ZF. To do this one must first clone the protein mRNA
> sequence from cDNA, then insert into an expression vector (I’m using
> PCS2+). However, whenever I clone and then sequence I never get 100%
> homology when blasting on NCBI. Although I use high-fidelity (Roche)
> enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa
> different from what is in the database.
> Is this normal? Is it a result of polymorphism? Or is it a problem
> with my methodology? And what degree of homology is needed (assuming
> no gaps exist) for the protein being expressed properly?
> Your help is appreciated!!
>
> Dr. Joanna Wilson
> Assistant Professor
> Department of Biology
> McMaster University
>
> 1280 Main Street West
> Hamilton ON
> L8S 4K1
>
> Tel: 905-525-9140 ext 20075
> Fax: 905-522-6066
> joanna.wil... from mcmaster.ca

Hi Joanna,

Thanks for the answer. The strange thing is that I do not get the same
‘errors’ when sequencing multiple clones. I can only think that this
is due to the fact that the cDNA was made from multiple larvae…? Or
that although I used HF the enzyme is causing errors. In any case,
when I use blastx for protein sequence (part of the protein – 900bp) I
get something like this:
 Score =  446 bits (1147),  Expect = 1e-123
 Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221
(0%)
 Frame = +2

(One alanine substituting a threonine)
Is such a substitution a problem?
Thanks very much
E.A.