T7 RNA polymerase for SP6 promoter?

[sobored]

T7 RNA polymerase for SP6 promoter?

hi guys!

i don't know how many molecular biologists are here, but i try my luck anyway
my question is,
is that possible using T7 RNA polymerase for transcribing genes under the control of SP6 promoter?


hopes for replies!
thanks a lot!

 

[Moonbear]

I assume you're talking about something you've inserted into a plasmid and your target sequence is properly inserted between the SP6 and T7 promoter sites. In that case, you'll have your sense DNA running in one direction and antisense in the other. For example, if you are trying to get antisense by transcribing in the SP6 to T7 direction, you'll get sense if you transcribe in the T7 to SP6 direction. So, no you don't get the same thing.

If you are thinking of some other context than what my answer covers, please tell us more

 

[sobored]

hi moonbear,

the story goes this way; we have the luc gene which lies between the SP6 promoter and the T7 promoter. the SP6 promoter lies in the start position of the gen while the T7 promoter lies in the end position of the gene. we wanted to transcribe the luc and then measure the activity of it. we did two tests. one of them we used SP6 RNA polymerase and the other we used T7 RNA polymerase. when we measured the luc activity we got activity in both tests, which mean the T7 RNA polymease did transcribe the gen by using the SP6 promoter, since the start of the gene lies downstream of the SP6 promoter. the T7 RNA polymerase could hardly bind to T7 promoter and transcribes, since this promoter lies in the end of the gene. so does it mean that T7 RNA polymerase can bind to SP6 promoter and transcribe the gene? okey, i hope you get my point...thanks!

 

[Moonbear]

Did you confirm that your plasmid is linearized and the enzymes are cutting in the correct place? I don't think that should cause this, but just ruling out stuff.

What do you know about the source of your luc gene? Is it possible that your insert has a T7 promoter in it? Have you had it sequenced in both directions?

Hopefully you already ruled this out, but have you tried this with a fresh vial of T7 polymerase? Just to be sure someone hasn't contaminated it with SP6? Stranger things have happened.

I've never heard of this happening, and it sure would mean a lot of trouble for those of us who rely on those promoters being different, though, if you do find this is really what's happening, or if anyone else here knows of similar things, I'd really like to know too...it might make me rethink my plasmid choices.

Another thing to do is check with the manufacturer of your vector and ask them about this problem. Every once in a while, something gets past quality control. If you can't find anything in your insert or in your technique that can account for this, have the vector sequenced just to be certain (or have the manufacturer check this).

I've also heard of "leaky" genes in transgenic mice - genes that are supposed to be regulated by a promoter, but are expressed at low constitutive levels. But, that gets well outside my realm of molecular biology knowledge, so I don't know the reasoning on that or if this could account for any of your problems with this particular gene.

 

[Sho'Nuff]

SP6 and T7 are names for bacteriophages (viruses that infect bacteria)
The SP6 polymerase is the polymerase used by the SP6 phage to make viral RNA once it infects a host (in this case salmonella) and the T7 polymerase is the one used by the T7 phage once it infects E.coli. Both RNA polymerases can bind to promotors and generate RNA. Of course the SP6 RNA polymerase has evolved to bind effectively to the SP6 promotors and will therefore have less affinity for a T7 promotor and vise versa. But using different polymerases with different promotors is generally possible.

 

[sobored]

thank you so much! this is to great help

 

[pattisyang]

SP6 and T7 are names for bacteriophages (viruses that infect bacteria)
The SP6 polymerase is the polymerase used by the SP6 phage to make viral RNA once it infects a host (in this case salmonella) and the T7 polymerase is the one used by the T7 phage once it infects E.coli. Both RNA polymerases can bind to promotors and generate RNA. Of course the SP6 RNA polymerase has evolved to bind effectively to the SP6 promotors and will therefore have less affinity for a T7 promotor and vise versa. But using different polymerases with different promotors is generally possible.

Dear Sho'Nuff,

Can SP6 promoter transcripe inserted fragment in eukaryotic cells like HepG2, Huh7? There is this pGEM-HBV vector constructed by inserting 1.3mer HBV genome into AatII & SmaI following a SP6 promoter. The author claimed that without strong promoter like CMV, this HBV genome was regulated only be the endogeneous promoter. If this is the case, then SP6 promoter must be totally ineffetive in eukaryotic cell lines, isn't it?

I would be more than glad if you could send me an email (pattisyang AT gmail.com), thus maybe we can be friends:)