Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus.
A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.
R L
RL
Allen
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Olins
J M
JM
Harp
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DE
Olins
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000032:analysis* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D056890:Eukaryota / Q000032:analysis* / Q000648:ultrastructure; D008793:Metrizamide; D008854:Microscopy, Electron; D013194:Staining and Labeling
D002843:Chromatin; D008793:Metrizamide