Replicating yeast isolated vectors in E. coli

Tritech Research Tritech_Research at lamg.com
Wed Nov 6 15:33:54 EST 1996


I am currently doing biochemical analyses of plasmids transformed into
yeast and I double check these results by isolating these plasmids and
retransforming them into E. coli.  I was wondering if anyone had a
"feeling" as to whether the plasmids recovered from yeast would be
"faithfully" replicated in E. coli.  These vectors are pUC-based with a
yeast 2micron and TRP1 gene.  I have heard conflicting views, and was
wondering if anyone may have some insight into this.  ANY comments,
etc., are appreciated!!

Kathy Baynton
----------------
The answer is "not always / it depends".  It will depend on the source
of the DNA cloned in yeast.  There are many eukaryotic sequences (my
specific experience is with C. elegans sequences), that are toxic and or
overly recombinagenic in E. coli, that are maintained just fine in yeast.
This is one of the reasons for the popularity of YAC cloning / libraries.
When you try to transform these plasmids into E. coli, you will get
rearranged versions, or nothing at all.

Excluding these "unclonable" sequences, there is some level of mutation
that can occur during/after the switch to E. coli, but that should be very
low if the DNA is handled carefully and the E. coli strain has its
recombination / DNA repair systems deactivated.

Andy Papp
TRITECH_RESEARCH at LAMG.COM




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