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reporter genes

Kathy Wilhelm wilhelmk at studentw.msu.edu
Thu Oct 6 15:16:00 EST 1994


> We are investigating a gene in Arabidopsis that is downregulated by light
> (minimal detection after 8h light). We were wanting to fuse several portions
> of the promoter to a reporter gene and assess their ability for light 
> responsiveness.  The presumed problem with gus is the stability of the protein 
> (50h in protoplasts according to Jefferson 1987). Has anyone tried a similar 
> experiment using histochemical analysis of gus or do we need to assess the activity
> of the promoter regions at the mRNA level? Or does anyone have a suggestion
> of a different reporter gene that may be useful in this situation?
>  Thankyou for your help.
.
Hi Susan,
.
We've been using gus both for promoter studies & for a mutant screen with 
a cold regulated gene.  A quick empirical test showed that it took about 
a week before gus activity was undetectable after moving from cold (inducing)
to warm (noninducing) conditions.  Other experiments had shown that the 
promoter shuts off within an hour and that the mRNA of gus is 
not very stable.  We were using the MUG assay for that, 
rather than X-gluc, which may or may not make a difference.  Also be aware
that there will be variation in the staining from plant to plant.  We used
histochemical staining to get a feel for localization of the message, but 
northerns to determine the effectiveness of promoter elements (Baker et al,
Plant Molecular Biology 24: 701-713).  If you want to avoid RNA, you might
want to look into luciferase (firefly or bacterial), which is said to be 
less stable.
.
Best wishes!
.
                                              Your fellow servant,
                                                 Kathy_|_
                                                       |
                                               Grad student, MSU
                                               wilhelmk at studentw.msu.e



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