Question about pBR322 and pUC18

[cmi...@channel1.com]

I have a question regarding these old standard vectors. I have searched
in the literature for my answer, but cannot find exactly what I seek. I
know that the difference between the two vectors (with respect to copy
number) is that pUC18 was created by deletion of the rop and a
single-base mutation in the origin of replication. This increased the
copy number from ~20 per cell to ~700 per cell. I am trying to find out
what the copy number of pBR322 would be if the rop was deleted, but the
origin of replication did NOT have the single base mutation. I would
suspect that this "intermediate" plasmid would have a much higher copy
number than pBR322 but would be lower than pUC18. I would also suspect
that it would be closer to pUC18 than pBR322 with respect to copy
number. I would welcome any ideas or insight (or references!).

Thanks!

Chuck Miller
cmi...@channel1.com

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[tfitz...@nexstar.com]

In article <7n2i9b$ch6$1...@news1.channel1.com>, Charles Miller
<cmi...@user1.channel1.com> writes

>I have a question regarding these old standard vectors. I have searched
>in the literature for my answer, but cannot find exactly what I seek. I
>know that the difference between the two vectors (with respect to copy
>number) is that pUC18 was created by deletion of the rop and a
>single-base mutation in the origin of replication. This increased the
>copy number from ~20 per cell to ~700 per cell. I am trying to find out
>what the copy number of pBR322 would be if the rop was deleted, but the
>origin of replication did NOT have the single base mutation. I would
>suspect that this "intermediate" plasmid would have a much higher copy
>number than pBR322 but would be lower than pUC18. I would also suspect
>that it would be closer to pUC18 than pBR322 with respect to copy
>number. I would welcome any ideas or insight (or references!).

and
>I have a question regarding the copy number of pBR322 based plasmids.
>I have looked for references in the literature, but cannot find the
>specific answer I seek. I know that the main differences between pBR322
>and pUC18 (with respect to replication and copy number) is that
>pUC18 was created from pBR322 by deletion of the rop and mutation in the
>ori. From Current Protocols and Maniatis, I have found that the rop
>is responsible for keeping the plasmid replication from going "nuts".
>As a consequence, the copy number of pBR322 is ~10 copies per cell while
>the copy number of pUC18 is ~700 copies per cell. The mutation in the ori
>is also a contributing factor in this higher copy number. I would like
>to know what the expected copy number would be of pBR322 WITHOUT the rop
>but with no mutation in the ori? I would expect that it would be much
>greater than pBR322 but less than pUC18. How much of a role does the
>mutation in ori play? Idea, answers, and references would be greatly
>appreciated!!

pMB1 plasmids deleted for rop, such as pAT153 (Twigg, A. J. and Sherrat, D.,
Nature 283, 216-218 (1980), derived from pBR327, have a copy number
approximately
3 times higher than the parent plasmid. pUC18, with a co-resident compatible
plasmid expressing rop,
has pBR322 copy number. ColE1 plasmid mutants generated by the Polisky lab,
with copy numbers 3-4 times higher than pUC18, are all reduced to wild-type copy
number
(15-20 copies in my hands) in the presence of a co-resident plasmid expressing
rop.
A single exception is pEW2705 (which has two mutations in the same stem as the
pUC mutation)
which is completely resistant to rop.

Tim Fitzwater
NeXstar Pharmaceuticals