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

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Summary

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 2M 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.

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Authors and Affiliations

  1. Istituto di Anatomia Umana Normale, Universitá di Ferrara, via Fossato di Mortara 66, I-44100, Ferrara, Italy

    L. M. Neri & S. Capitani

  2. Istituto di Citomorfologia Normale e Patologica del C.N.R., c/o Istituto Ortopedico Rizzoli, via di Barbiano 1/10, Bologna, Italy

    L. M. Neri, N. M. Maraldi & C. Cinti

  3. Istituto di Anatomia Umana Normale, Universitá di Bologna, via Irnerio 48, Bologna, Italy

    G. Mazzotti & A. M. Martelli

  4. Oncologia Ortopedica, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, Bologna, Italy

    N. Baldini

  5. Istituto di Morfologia Umana Normale, Universitá di Chieti, via dei Vestini, I-66100, Chieti, Italy

    R. Rana

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  1. L. M. Neri
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  2. G. Mazzotti
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  3. S. Capitani
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  4. N. M. Maraldi
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  5. C. Cinti
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  6. N. Baldini
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  7. R. Rana
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  8. A. M. Martelli
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Neri, L.M., Mazzotti, G., Capitani, S. et al. Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine. Histochemistry 98, 19–32 (1992). https://doi.org/10.1007/BF00716934

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  • DOI: https://doi.org/10.1007/BF00716934

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