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Protoplast problems

Tue Jan 18 05:17:16 EST 1994

Dear Plant Net People,

	I wonder if there is anyone out there who can help.
	As some of you may have read in Plant Physiology (Pyke & Leech 91,92,94)we at York have isolated some mutants of Arabidopsis with abnormal numbers
and sizes of chloroplasts. Some of these chloroplasts are of considerable sizes
(c2500um2 plan area compared to 50um2 in wild type).
	We have two 3rd year project students working on characterising the
proteins in the mutants and they hope to isolate proteins from intact isolated
chloroplasts. As you can imagine the "intact" part of this idea is a bit of a
problem with the very large chloroplasts due to their size and large starch
grains. The largest chloroplasts (those of mutant "arc6") appear to wrap 
themselves around the cytoplasmic compartment, so to speak, producing a 3D
structure around the cell. These chloroplasts (often only one chloroplast per
cell compared to 80 in wild type) therefore may have a plan area which is
larger than the cell itself.

	So, my questions are:-
a) Does anyone have any experience isolating large chloroplasts (with starch
	grains) from any plants which leave them intact?
b) If we destarch the chloroplasts by leaving them in the dark, how much will
	this affect the proteins of the envelope, thylakoid and stroma?
c) Does anyone have a protoplast prep (possibly from Moss?!) which involves
	a minimum of centrifugation
d) Broken protoplasts and arc6 intact chloroplasts will possibly be about
	the same size. Will it be possible to separate them (eg through a 
	sucrose cushion). Any ide

A bit of a long one, that. I thank everyone in advance for any help you may 
be able to offer.
Steve Rutherford

Dept. Biology,
University of York,
York YO1 5DD. UK.


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