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Is this gene going to express? - is only the firs ATG read and express or all 3 reading frame? (Jan/10/2008 )

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Hi everyone!!! smile.gif
I need your opinion...!!!!!

We cloned a gene for expression, directed by the CMV promoter.
We digest the vector with EcoRV and the gene (previously cloned) with SnaBI and ligate.
We didn’t realize that an ATG is created 5 bases before the gene-ATG because of the union of EcoRV and SnaBI ends.

Do you think that just the first ATG is going to be read and the real gene is not going to express ??

Bye the way, after the first ATG there would be a codon stop.

I’m attaching the sequence.

Thanks in advantage!

ATG created:
atg taa tgt tgg gga..
ATG stop

Right ATG:
atgta atg ttg gga tgc ttg
Met Leu Gli..(right gen)

-aztecan princess-

I think your transgene with be expressed. But one has to try it out to confirm.

-scolix-

Unfortunately, there is not antibodies available, what do you suggest?

Here, some people says the machinery recognize and take ONLY the fist ATG, so,our construction would result in a Met and that would be all. Other says the 3 reading frame are always read, and could result in different peptides (if it’ll possible), some of them could be useless and some or just one could be functional.

I need opinions!!!

-aztecan princess-

they are very close so i guess it will ber expressed.
A semi quantitative RT pcr should let you know if the RNA is increased.
second possibility is to do local mutagenesis or do other combination of comatible sites (if possible) by producing the insert from a PCR

-fred_33-

Its possible that your gene will be expressed in the wrong reading frame so mRNA analyses
might not confirm expression.
You have the sequence ATG ta ATG
There is no Kozak so transcription/translation may start at the first ATG and then the 2nd
ATG will be out of frame.
What I would do: Reclone the gene into a vactor that has a HA/FLAG/MYC tag.
Then you can do a Western. If you can't do a Western it is tricky to interpret results.

-mikew-

I would guess that there will be expression of the gene but the level might be lower because of the other start codon.

I would go with fred's suggestion to change the first ATG by pcr or mutagenesis.

-scolix-

Thank you very much for your comments!

There is a relatively easy way to re-clone the gene in the vector without the first ATG (the wrong one), since it is created when ligate EcoVI-SnaBI end ligation. So just started to do it.

Any way, I really wanted to know if the gene would be expressed or not cloned as it’s now because I wasn't sure. So thanks for your anwers! smile.gif

Other Question…. We are cloning viral genes for expression like this gene. This genes don’t have a native kozak sequence. Do you think we would add it to have a better expression, or they would be expressed without problem?

If the virus use the cell‘s machinery (eucariotic) to express their genes in nature, I think the kozak sequence won’t be necessary for express the same genes in an expression vector. But I think may be adding kozak sequence would increase the expression level.

What do you think????????

-aztecan princess-

i remeber once i asked me same question.
i can't find the thread, but i remember that the answer was that the kozack sequence is included in your expression system before initial ATG.
I checked on my expression vector (it was pIRES which uses CMV too) and i found it in the vector sequence.

-fred_33-

QUOTE (fred_33 @ Jan 11 2008, 04:57 PM)
i remeber once i asked me same question.
i can't find the thread, but i remember that the answer was that the kozack sequence is included in your expression system before initial ATG.
I checked on my expression vector (it was pIRES which uses CMV too) and i found it in the vector sequence.



But the kozak sequence include the initial ATG of the protein, not as Shine-Dalgarno Séquense In prokaryotes that is some bases before the ATG, how can the vector include a kozak sequence… specially the G after ATG??

kozak seq:
ACCATGG (gcc)gccRccAUGG

-aztecan princess-

The kozak sequence is not required for viral expression of the RNA, because as you said, the virus will use the cell's machinery to express the RNA. The advantage of the kozak sequence resides in the fact that it facilitates the translation of the RNA into the protein. You would therefore expect more protein to be synthetized if the kozak sequence is included.

-Madrius-

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