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Nucleic Acids Research, 1986, Vol. 14, No. 2 823-841
© 1986

Articles

Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus*

Carmen L. Cadilla, Joel Harp, John M. Flanagan, Ada L. Olins and Donald E. Olins

University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory Oak Ridge, TN 37831, USA

Received November 11, 1985. Accepted December 18, 1985.

Euplotes eurystomus la a hypotrichous ciliate containing a transcrip-tionally active nacronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner his tones, an Hl-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysls. Mlcroccocal nuclease digestion yielded an average nucleosome repeat length of 187 ± 25 bp for soluble chroaatin; and 190 ± 9 bp for isolated nacronuclei. Thermal denaturatlon profiles of MAC chromatin in 0.25 nM EDTA display two main transitions at about 76 and 83°C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 20 chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

*Research supported by NIH research grants GM-19334 to D.E.O., and to A.L.O. (GM-29429), by an NIGMS National Research Service Award to C.L.C. (GM-10079-02) and by the Office of Health and Environmental Research, D.S. Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.


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