title: Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.
abstract: BACKGROUND
  Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.
  
  
  RESULTS
  Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.
  
  
  CONCLUSIONS
  We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.
authors.affiliation: Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. mjbuck@buffalo.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Givens
authors.forename: Larry D
authors.initials: LD
authors.lastname: Mesner
authors.forename: Joyce L
authors.initials: JL
authors.lastname: Hamlin
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Buck
authors.forename: Joel A
authors.initials: JA
authors.lastname: Huberman
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