> min wrote:
>> Hi,
> I'd like to know whether there is any problem in cloning a gene in the
> same strain host in which that gene is mutated. --Cloning host must be
> recombination-deficient ? etc.
> Any replies would be appreciated.
> Min
> Seoul National University
>
It would depend on what type of mutation is present (leaky ie point
mutation, deletion, etc.). I know that in situations where the gene
being cloned makes a multimeric enzyme, the host gene may interfere b/c
it may also form an enzyme with similar properties (protein-protein
interactions are similar). For example:
E. coli dihydroorotase (DHOase) is a 40-kDa monomer that forms 80 kDa
dimers, and is encoded by the pyrC gene. When various forms of the
mammalian DHOase (also able to form dimers) were cloned into E. coli,
some interference was noted from the E. coli DHOase, which formed
heterodimers with the mammalian DHOase. This occurs with a very low
frequency, but when the assays are sensitive enough (radioisotope), they
can cause significant problems. This is noted in a paper by David R.
Evans sometime around 1995.
I would suspect that one's best hope is to clone into a strain with a
deletion of the gene of interest on the chromosome (I am doing that very
thing; I have a strain on order that has THREE deletions for the
different isoforms of the enzyme on the chromosome; never hurts to be
safe rather than sorry).
--
C. J. Fields
Graduate Student, Dept. of Biological Sciences
The University of North Texas
Denton, TX
email : cjfields at jove.acs.unt.edu
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