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Genetics as it applies to evolution, molecular biology, and medical aspects.

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Postby ericarebelo » Sat Feb 11, 2012 6:32 pm

Hi! I'm supposed to make a presentation about Recombinant DNA for Genetics. I have all figured out, except this one thing which is bothering me:

If the gene that was inserted into the vector contains a sequence that introns or contains signals which act as terminators to a bacterial host. This results in premature termination, and the recombinant
protein may not be processed correctly, be folded correctly, or may even be degraded.

My first question is : Does this happen only in bacterial cells ?
My second one is: Does this happen only in the translation process, or can it occur during transcription and replication ?

Please answer, ASAP
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Postby AstraSequi » Sun Feb 12, 2012 1:44 am

Hi there,

I'm not exactly sure I understand your questions, but I can mention a few things:

For your first question: the reason that these problems occur is because of the differences between the eukaryotic and bacterial systems, which I think you understand (so, for example, you would want to delete introns out of the DNA before inserting it in your vector). In a eukaryotic expression system, then these problems will not be as bad because you will have the machinery for splicing, glycosylation, etc in the cells. I imagine that you might get related problems in archaea as well, but there aren't any archaeal expression systems as far as I know.

For the second question: as above, any problem that occurs will be due to the difference between the systems. Your gene will only be expressed if it has an appropriate promoter, and this is usually different between species. Similarly, the plasmid must be able to replicate, so it needs an origin of replication, which is also different between species.

If you're asking about incorrect transcription or replication specifically because of the sequence, I would doubt it - the only difference you have is the DNA sequence of the specific protein, and both transcription and replication will continue until finished regardless of sequence as long as it is able to begin. (Of course, you will also need the correct signals to tell it to stop in the case of transcription.)

Also, you can get incorrect processing, incorrect folding, or degradation in any system that is not the same one that the protein comes from (and sometimes even in the same one, if the conditions aren't right). It's just that we maximize our chances of everything working correctly if we use a closely related system rather than a distantly related one (i.e. bacteria). Bacteria are usually the system of choice because of their fast replication, and we only go to other systems when it's clear that expression won't work in bacteria.

I hope that helps. :)
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