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A stop codon killed my ORF!

A stop codon killed my ORF! - Protocols and Methods Forum

A stop codon killed my ORF! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 03-05-2005, 05:36 PM
Emilie
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Default A stop codon killed my ORF!



Hello all,

After performing multiple RACE PCR experiments in order to obtain the
5'end of my gene, I finally succeded in obtaining the entire gene.
Everything seemed to be ok, the predicted peptide showed good
alignement to other related proteins with plenty of conserved regions,
but... there is a stop. About 600 bp downstream the ATG [total length
= 2.8 Kb] . There are conserved regions before and after the stop.
I checked the sequence several times, reamplifying with a
proof-reading polymerase as well as my usual one.
I am looking for a plausible explanation. Why the stop? Could it be a
pseudogene? why would it be transcribed?
Could it be a defect in splicing a small intron, resulting in the
stop?
I don't really know how to justify that result, can you help?
Any advice is grately appreciated!

-------
Emilie
Department of Biological Sciences, Warwick University
[Only registered users see links. ]
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  #2  
Old 03-13-2005, 08:05 AM
Dr Engelbert Buxbaum
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Default A stop codon killed my ORF!

Emilie wrote:


Which of the 3 stop codons? Amber can code for the unusual amino acid
pyrrolysine, opal for selenocysteine.
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  #3  
Old 03-14-2005, 08:34 AM
C.Emilie@gmail.com
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Default A stop codon killed my ORF!


I have a UGA (opal). Is it (selenocysteine) a frequent occurence. I
have never heard of it for our fungus...
I'll read more on that, thanks a lot

Emilie

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  #4  
Old 03-14-2005, 09:13 AM
Trond Erik Vee Aune
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Default A stop codon killed my ORF!



Dr Engelbert Buxbaum wrote:


All stop codons can if surrounding conditions is favorable, code for
recoding events (frameshifting, hopping, insertion of unusual codons etc.).

Opal is considered to be the least effective (in promoting translation
termination) stop codon of them all, and amber the most effective
(promotes translation termination with highest efficiency), at least in
in vitro experiments.

Trond Erik

--
Trond Erik Vee Aune
Department of Biotechnology
Norwegian University of Science and Technology
Email: [Only registered users see links. ]
[Only registered users see links. ]

- Must be sad being a dyslectic, agnostic insomniac, lying
awake during the night, wondering if there really is a dog
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  #5  
Old 03-14-2005, 09:20 AM
Trond Erik Vee Aune
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Default A stop codon killed my ORF!



[Only registered users see links. ] wrote:


Insertion of celenocysteine in eukaryotes is promoted by bSECIS elements
situated downstream of the opal codon. Close inspection of your gene
should be able to discover any bSECIS elements. If there's no bSECIS
element in your sequence, the decoding event must then be of another
kind than celenocystein insertion, or happening through a novel
mechanism that doesn't rely on bSECIS elements.

Have you looked at your protein on a protein gel (or western) to see if
the stop codon is efficient? You could also try to change the tga codon
to tag codon to see what happens.

Here's a reference to a great review of decoding events in both
prokaryotes and eukaryotes:

Namy et al. Reprogrammed Genetic Decoding in Cellular Gene Expression.
Molecular Cell. Vol. 13, 157-168. 2004.

Trond Erik


--
Trond Erik Vee Aune
Department of Biotechnology
Norwegian University of Science and Technology
Email: [Only registered users see links. ]
[Only registered users see links. ]

- Must be sad being a dyslectic, agnostic insomniac, lying
awake during the night, wondering if there really is a dog
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  #6  
Old 03-18-2005, 05:32 PM
Boney Tee
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Default A stop codon killed my ORF!

Trond Erik Vee Aune wrote:

Just to be clear.... the SECIS element would most likely be found in
the 3' UTR of your RNA of interest. What is your protein? Enzymes
involved in funny re-dox type reactions often do contain selenocyteine
at their catalytic centers.

T
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  #7  
Old 03-19-2005, 12:23 PM
C.Emilie@gmail.com
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Default A stop codon killed my ORF!

I have a lipoxygenase. Hoperfully I'll find something in the 3'UTR that
will help understand this stop disrupting such a nice ORF!

E.

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