Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus.
Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-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 proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees 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. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.
Cadilla
C L
CL
Harp
J
J
Flanagan
J M
JM
Olins
A L
AL
Olins
D E
DE
D000818:Animals; D002460:Cell Line; D002645:Chickens; D002843:Chromatin / Q000302:isolation & purification / Q000648:ultrastructure*; D016798:Ciliophora / Q000235:genetics / Q000648:ultrastructure*; D002942:Circular Dichroism; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000648:ultrastructure; D008836:Micrococcal Nuclease; D008854:Microscopy, Electron; D009691:Nucleic Acid Denaturation; D014158:Transcription, Genetic
D002843:Chromatin; D004247:DNA; D008836:Micrococcal Nuclease