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Third-party-funded project

Title:
Functional organization and assembly of the nucleolus

Project management at the University of Würzburg:

Participating scientists:

Abstract:
We have expressed a series of different nucleolar proteins fused to GFP or its spectral variants in cultured mammalian cells. We now plan to analyze dynamic properties of nucleolar structures and of constituents of the transcription machinery (RNA polymerase I and transcription factors) using photobleaching techniques and to observe structural rearrangements in relation to rDNA gene activity in living cells. In addition, we have successfully expressed fusions of GFP and ribosomal proteins which are apparently correctly incorporated into nucleolar preribosomes and exported as mature subunits to the cytoplasm. We will immunopurify preribosomes from the nucleoplasmic fraction of transfected human cells using immobilized anti-GFP antibodies. The ability of the purified preribosomes to enter the nuclear export pathway will be examined by fluorescence microscopy after microinjection into nuclei of normal cells. The biochemical composition of the export-competent preribsomes will then be analyzed by gel electrophoresis and mass spectrometry. Using this assay, the identification of factors mediating ribosomal export in mammalian cells should become possible

Key words:
    GFP fusion proteins
    photobleaching
    nucleolus
    ribosomes
    nuclear export

Projekt period: from 04.2000 to 03.2002

Funding institution:
DFG ( Sche 157/12-2 ) ,Granting date: 28.02.2000

Preceding project:
DFG ( Sche 157/12-1 )