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

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

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)823-841
Number of pages19
JournalNucleic acids research
Volume14
Issue number2
DOIs
StatePublished - Jan 26 1986

All Science Journal Classification (ASJC) codes

  • Genetics

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