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ORI names


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11 replies to this topic

#1 Ikar

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Posted 16 November 2010 - 02:49 PM

Hi,

I have a question about the different kinds of the "origins of replication".

I know that the sequence for a autonomous plasmid in yeast (S.pombe) is called "ARS".
But how is it called in other organisms?
For example S.cerevisiae?

I think that in E.coli this plasmid ori is called "oriT"?

I hope you can understand my question and will be able to help me!

br,
Ikar

#2 HomeBrew

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Posted 16 November 2010 - 05:15 PM

In the bacterial plasmid world, "oriT" indicates the origin of transfer in a promiscuous plasmid. "OriV" is the plasmid's origin of vegetative growth (aka replication). All plasmids will have an oriV, as the all must replicate, but not all will have an oriT, as not all are transmissible.

#3 Ikar

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Posted 17 November 2010 - 01:31 AM

Thank you for this expeditious answer.

I think it's a typing error and you mean "OriT" instead of "OriV" in the last part of your last sentence?

I hope you might forgive the unknowing student if he asked you: "Does all bacteria have the same DNA-sequence in the OriV?
Or does every bacterium have his own individual OriV-Sequence?"

Edited by Ikar, 17 November 2010 - 01:32 AM.


#4 HomeBrew

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Posted 17 November 2010 - 04:30 AM

I think it's a typing error and you mean "OriT" instead of "OriV" in the last part of your last sentence?


Yes, you're right. I've fixed the typo.

Does all bacteria have the same DNA-sequence in the OriV? Or does every bacterium have his own individual OriV-Sequence?


Are we still talking about plasmids? If so, then the answer is no -- not all bacteria have a plasmid, so those without a plasmid would not have a plasmid-borne OriV. Moreover, the exact sequence of the oriV varies from plasmid to plasmid.

If you mean on the chromosome, each bacterial chromosome has a single origin of replication -- in E. coli, this is called the oriC. Archaea have several origins of replication along their circular chromosome, and Eukaryotes usually have multiple origins of replication on each of their linear chromosomes.

#5 Ikar

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Posted 17 November 2010 - 04:23 PM

Excuse me for my imprecise explanations. I am from Germany and do not often use english language so I have my difficulties to illustrate something.
But that is also the reason why I try using an english forum: to improve my language skills while I get the answers to my questions ;)

Maybe it is easier for me if I start anew:

A short summary on what I've understood:
- E.coli has on his chromosome a oriC for replication of its genome
- It has a oriV on a plasmid for its autonomous replication (the replication of this plasmid)
- each type of bacterium has it's own kind of oriC
- if the species has plasmids, the oriV sequence will be a bit different between the species

But if you transform a vector plasmid in any kind of bacterium, do you always use the same oriV which is located on this
plasmid for its replication?
Or do you use different kinds of "oriV"s on your vector plasmids for each species?

Edited by Ikar, 17 November 2010 - 04:26 PM.


#6 HomeBrew

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Posted 17 November 2010 - 05:33 PM

Some plasmids replicate in multiple species -- these are known as broad host range plasmids. Other plasmids only replicate in a single species or in a small handful of closely related species. There are engineered plasmids that contain two oriVs -- one that operates in one species and a second one that is functional in another species -- these are called shuttle vectors, and allow one to do things like cloning manipulations in E. coli and then move the completed construct into another genus, like Bacteroides, or even another kingdom, like fungi.

#7 Ikar

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Posted 18 November 2010 - 01:59 AM

Thank you. I think I got it.

When I am going to use such a shuttle vector e.g. in E.Coli and S.cerevisiae, then this plasmid should contain
an ARS (replication in the yeast) and a oriV (replication in E.Coli)!?

#8 HomeBrew

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Posted 18 November 2010 - 04:58 AM

Yes. In addition, the plasmid needs a selectable marker for each organism. In E. coli, this is usually an antibiotic resistance gene (ampicillin, tetracyline, kanamycin, etc.), and in yeast selection is usually made by using a shuttle vector that complements an auxotrophic mutation in the recipient yeast strain (ura3-52, his3-D1, leu2-D1, etc.). See here for examples.

#9 Ikar

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Posted 18 November 2010 - 11:03 AM

Thank you. I think my question is perfectly answered!

#10 Ikar

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Posted 21 November 2010 - 03:57 AM

While reading the marvelous homepage you gave me (http://dbb.urmc.roch...yeast/Cont.html)
I faced another problem or rather question:

If I transform a plasmid (with an ORI) into a yeast cell the plasmid will replicate once per cell cycle.
How can I be sure that the daughter will get one of this two plasmids?
Therefore I need to integrate a CEN-DNA-Sequence in the plasmid, so the mitotic spindle
can be attached.
-> That is only a consederation of me. I would be happy if you could tell me whether I'm right or wrong!

br,
Sebastian

#11 HomeBrew

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Posted 21 November 2010 - 05:47 PM

Now you've moved beyond me. I don't work with yeast, but I'm sure someone else on the forum who knows more about yeast genetics will jump in...

#12 Ikar

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Posted 22 November 2010 - 01:55 AM

That's a pity, but I have to thank you a lot for your quick and helpful answers nonetheless!
You were a great help for me.

Now I am hoping that someone else can answer my question - hopefully with the same
quality you were doing. :)

br,
Sebastian




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