> >This is very interesting. Two questions:
> >How did you determine protein concentrations in the pollen tubes?
>
>I did not! What I meant to say was that brightly fluorescent pollen tubes
>were affected, whereas weakly fluorescent tubes could grow normally
>(different bombarded pollen tubes on one particular plate express the
>transferred genes at variable levels and emit fluorescence of variable
>intensity).
>
>The promoter I use is known to confer strong gene expression in pollen
>tubes. Also, the fluorescence I get is really bright!

CFP, in my hands, is a lot less bright than EGFP or YFP. If you get
equal brightness with CFP, that might indicate that you have much
higher overexpression. (I know you would expect equal expression
levels for constructs under the same promoter, but still...)

>
> >Did you filter out the excitation wavelengths of CFP as a control to
> >see whether the effect is not due to CFP mediated phototoxicity?
>
>
>It does not look like the growth defects were caused by phototoxicity.
>Affected pollen tubes have swollen tips, which are visible the moment you
>switch on the epifluorescence illumination. The tip swelling appears to
>result from abnormal growth over some time in the absence of epifluorescence
>illumination. I suspect, that the CFP toxicity we find in pollen tubes may
>have something to do with the observation made by Heim et al. (1994, PNAS
>9,12501) that GFP carrying the Y66W mutation (which is responsible for CFP's
>fluo characteristics) is only inefficently expressed in E. coli (in contrast
>to other GFP versions). If that should be the case, it maybe difficult to
>get rid of the problem!

It seems rather unlikely that a point mutation at the inaccessible
core of a protein which does not seem to screw up folding has a
pronounced effect on toxicity of the protein. There is a number of
possibilities why this could still be the case, but I still think
that the increase in toxicity most likely would be due to an increase
in the generation of radicals. Long-term illumination with normal
light might do it, there is no need for epifluorescence illumination.
Anyway, all of this is completely speculative until the relevant
controls have been done.

Just my $0.02


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