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Cosmid vectors

Toni Schaeffner schaeff at lmb.uni-muenchen.de
Wed Mar 20 06:14:05 EST 1996


Dear netters,

Subject: Answers to my inquiry about cosmid vectors suitable for subcloning
YACs other than pBIC20 and pLZO3.

Altough by accident I already posted a preliminary summary of answers, here
is the complete collection of letters I got. At a few points I introduced
comments or cross-references).

Tony Schaeffner, Munich, Germany

________

I can provide pOCA18 or pOCA28 if you need them.

Inserts are very stable in pOCA.  It was built for stability.  See: Nucleic
Acids Research 16:10765 (1988) for an analysis of the stability of an
arabidopsis library in pOCA18.

Cloning capacity of pOCA18 15-20 kb.

pOCA18 & 28 carry kanamycin plant markers (NOS:NPTII gene).  This marker works
well for my lab with arabidopsis, tomato, tob.


pOCA18 has the disadvantage that the bacterial marker is tetracycline
resistance.  The A136 chromosomal background (most nopaline strains) give rise
to spontanious resistance at a frequency of 10-5 (see the NAR paper).  This can
be worked around but I made pOCA28 to solve this problem.  The marker in pOCA28
is strep/spec.

Best wishes,
Neil Olszewski

_____________

We have used the pOCA vectors and cosmid library prepared by Neil Olszewski
to clone a selection of Arabidopsis genes. He can be reached at the
Dept. of Plant Biology, University of Minnesota, on FON 612 625 3129 or FAX
612 625 1738

Cheers

Malcolm Bennett
Dept. of Biological Sciences
University of Warwick
Coventry
UK

_______________________________

I don't like poca, because it is high copy, and that seems like a fundamentally
bad idea to me.  Brian Staskawicz uses a vector made by Jonathan Jones
called pCLD04541, which is Tc in bacteria, and Km in plants.  They have
made several libraries from YACs now (they make a library from the whole yeast
strain that carries the YAC), and so far have not found any gaps in their
libraries.  I also made a library in this vector, and the person who screened
it said it was complete for the YAC.
The only trouble is that the Agrobacterium strain normally used, GV3101,
mutates to Tc resistance at high frequency.  The story from Brian's lab is
that to select Agro carrying the cosmids, select with Tc at 2ug/ml, and the
small colonies are the right ones.  Large colonies, or colonies that appear
at Tc concetrations higher than 2, are spontaneous mutants.  This sounds nasty
to me, but apparently they get it to work just fine.  I havn't tried the
Agro transformation part myself, yet.
The vector is proprietary, you have to get it from Jonathan, but it's no
problem.  He also has somme derivatives for Basta, but as of six months ago,
these had not actually been used for Arabidpsis trasformation, so I was
hesitant to use them  They may have the same problem with Tc, I don't know.
If you decide to use pCLD04541, it might be worthwhile chatting with
Doug Dahlbeck in Brian's lab, to make sure you have the right story about
the Tc resistance.
I think I have POCA28 somewhere, if you want it.
Jane   (Glazebrook, MGH, Harvard)

Reply:
In reply to Jane's concerns about the copy number.  I find the high copy
number to
be an advantage in terms of preparing DNA and have seen no evidence of
instability in E. coli.

Best wishes,
Neil

Comment: As written also below, actually Clare Lister constructed pCLD04541
(Tony)

______________

We have used the pOCA vectors and cosmid library prepared by Neil Olszewski
to clone a selection of Arabidopsis genes. He can be reached at the
Dept. of Plant Biology, University of Minnesota, on FON 612 625 3129 or FAX
612 625 1738

Cheers

Malcolm Bennett
Dept. of Biological Sciences
University of Warwick
Coventry
UK

_______________________

While cloning RPM1 resistance gene in Jeff Dangl's lab, we used pCLD04541
from Jonathan Jones' lab to clone our yacs of interest. It is a cosmid
vector suitable for transformation where we inserted 15 to 20 kb of yeast
DNA.In fact we  did a complete yeast library containing our yac in this
vector , and then selected the interesting clones with a probe made out of
the yac isolated by PFGE. It worked quite well, and we could have the RPM1
gene by complementation analysis this way.
Some references : Transgenic  Research 1, 285-297 (1992) for the vector
and Science 269, pp 843-846 (1995) for cloning RPM1.

Sincerely,
Laurence Godiard

Laurence Godiard
Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes
UMR CNRS/INRA 05
BP 27
31326 Castanet-Tolosan Cedex, France.

__________________

We are in the process of writing a paper that includes the use of cosmid vector
04541 (constructed by Clare Lister in Caroline Dean's lab) for subcloning YACs.
You should remember my talking about it in the Genomics workshop you organised
in Madison.  The insert size range is 10 to 24 kb and in-planta selection
Kanamycin resistance.  Like pOCA18 it is based on pRK290, so clones are very
stable.  The vector has been used successfully for complementation of mutant
phenotypes in Arabidopsis.  The vector and protocol have been sent to some
labs, but I have not heard back from anyone.  I can routinely make 30- to 60-
fold redundant libraries and have so far always obtained complete coverage.
Yours sincerely,
Ian Bancroft, John Innes Centre, Norwich

_________________

> The only trouble is that the Agrobacterium strain normally used, GV3101,
> mutates to Tc resistance at high frequency.

We've been able to circumvent this problem by using Kan50 to select for
transformants with pCLD04541.  Apparently the 35s promoter is active
enough in Agro. for this to work.

mcdowell at email.unc.edu (John M. McDowell)

_____________________________

Clare Lister (clare.lister at bbsrc.ac.uk), who actually constructed pCLD04541,
wrote concerning the spontaneous tet resistance:

    I am not actually the person who does the
transformations in Caroline's lab so I've never put
any pCLD04541 clones into Agro! However I talked to
Tania Page who does and she said that she does not
use pGV3101. She did actually try it when everyone
was saying how well it worked for vacuum
infiltration. She said she didn't get as many
colonies as the strain she had been using
previously and hadn't seen any spontaneous tetR
colonies. She has reverted back to using pGV2260
and said it works well. If you have any further
questions about it her e-mail address is:-
paget at bbsrc.ac.uk
______
Comment:

In a recent paper in Cell (Jan or Feb 1996) the Jones lab used pCLD04541 to
construct a tomato genomic library in E. coli SURE (TM) and later on
LBA4404 as Agrobacterial strain for (successful) complementation (Cf-2
resitance locus).
Tony
_______________________________________

I constructed a cosmid vector based on the vector described by  (Bouchez,
D., Camilleri, C. and Caboche, M. (1993). A binary vector based on Basta
resistance for in planta transformation of Arabidopsis thaliana. C. R.
Acad. Sci. Paris 316, 1188-1193), used for the initial infiltration
experiments. It has a BamHI cloning site and a BASTA-R gene that allows
easy screening for transformants in soil. The vector is very stable in
Agrobacterium.  The insert size is about 20 kb and the transformation into
Arabidopsis by infiltration worked fine.

Herman Hofte
Laboratoire de Biologie Cellulaire
INRA
Route de Saint-Cyr
78026 Versailles cedex
Tel: 33-1-30833390    Fax: 33-1-30833099



*   Dr. Tony Schaeffner
*   Institut f. Biochemie
*   Ludwig-Maximilians-Universitaet
*   Wuermtalstrasse 221
*   D-81375 Muenchen, Germany
*   Tel. (0)89-7401-7311
*   Fax. (0)89-7401-7314
*   email: schaeff at lmb.uni-muenchen.de





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