Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine

L. M. Neri, G. Mazzotti, S. Capitani, N. M. Maraldi, C. Cinti, N. Baldini, R. Rana, A. M. Martelli

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The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of lfuorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.

Original languageEnglish
Pages (from-to)19-32
Number of pages14
Issue number1
Publication statusPublished - Aug 1992

ASJC Scopus subject areas

  • Anatomy


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