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Vol. 15, Issue 4, 1816-1832, April 2004

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NO66, a Highly Conserved Dual Location Protein in the Nucleolus and in a Special Type of Synchronously Replicating Chromatin

Jens Eilbracht ^*, Michaela Reichenzeller ^*, Michaela Hergt ^*, Martina Schnölzer , Hans Heid ^*, Michael Stöhr , Werner W. Franke ^*, and Marion S. Schmidt-Zachmann ^* 

^* Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany; Protein Analysis Facility, German Cancer Research Center, D-69120 Heidelberg, Germany; and Cytometry Facility, German Cancer Research Center, D-69120 Heidelberg, Germany

Submitted August 25, 2003; Revised December 17, 2003; Accepted January 8, 2004
Monitoring Editor: Joseph Gall

It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.

Abbreviations used: aa, amino acid(s); AMD, actinomycin D; DFC, dense fibrillar component; FACS, fluorescence-activated cell sorting; FC, fibrillar center; FITC, fluorescein isothiocyanate; GC, granular component; HSP, high-speed pellet; HSS, high-speed supernatant; LSP, low-speed pellet; MALDI, matrix-assisted laser desorption ionization; SMN, survival of motor neuron; PCNA, proliferating cell nuclear antigen; PML, promyelotic leukemia.

Online version of this article contains supplemental figures. Online version is available at www.molbiolcell.org.

Corresponding author. E-mail address: m.schmidt-zachmann{at}dkfz.de.

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