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CHEMICALLY INDUCED ECDYSIS AND ISOLATION OF NUCLEI FROM THE DINOFLAGELLATES GONYAULAX POLYEDRA AND GONYAULAX TAMERENSIS
Author(s) -
Morris R. L.,
Rizzo P.J.
Publication year - 2000
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1046/j.1529-8817.1999.00001-147.x
Subject(s) - ecdysis , biology , spheroplast , dinoflagellate , centrifugation , isolation (microbiology) , biochemistry , botany , microbiology and biotechnology , escherichia coli , moulting , larva , gene
Armored dinoflagellates such as Gonyaulax sp. are not good candidates for isolation of intact clean nuclei, due to difficulty in breaking the cells. Previously, isolation of nuclei from Gonyaulax was achieved using vigorous cell disruption by ultrasonication (Rizzo & Hastings, unpublished observations). A detergent method for production of spheroplasts from Gonyaulax was published some time ago (Adamich & Sweeney, 1976), but was not reproducible (Rizzo & Hastings, Morris & Rizzo, unpublished observations). We describe here a modification of the method described by Adamich & Sweeney that is highly reproducible. Ecdysis and production of intact dinoflagellate spheroplasts with a yield of virtually 100% within 5 minutes, has been achieved through the use of anionic and nonionic detergents in hypotonic buffers. This method of ecdysis followed by a gentle spheroplast breakage in a hypotonic buffer allowed the release of nuclei for later biochemical analysis with minimal damage or breakage. The nuclei are subsequently purified by centrifugation in a Ficoll discontinuous gradient.