Abstract
Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G1/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 μm in all S phase cells except those at G1/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.
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References
Adolph, K.W.: Isolation and structural organization of human mitotic chromosomes. Chromosoma (Berl.) 76, 23–33 (1980)
Adolph, K.W., Cheng, S.M., Laemmli, U.K.: Role of nonhistone proteins in metaphase chromosome structure. Cell 12, 805–816 (1977)
Amaldi, F., Carnevali, F., Leoni, L., Mariotti, D.: Replicon origins in Chinese hamster cell DNA. I. Labeling procedure and preliminary observations. Exp. Cell Res. 74, 367–374 (1972)
Aula, P.: Electron microscopic observations on Sendai virus-induced chromosome pulverization in HeLa cells. Hereditas (Lund) 65, 163–170 (1970)
Benyajati, C., Worcel, A.: Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster. Cell 9, 393–407 (1976)
Berezhey, R., Coffey, D.S.: Identification of a nuclear protein matrix. Biochem. biophys. Res. Comm. 60, 1410–1417 (1974)
Berezney, R., Coffey, D.S.: Nuclear protein matrix association with newly synthesized DNA. Science 189, 291–293 (1975)
Campbell, C.E., Worton, R.G.: Chromosome replication patterns in an established cell line (CHO). Cytogenet. Cell Genet 19, 303–319 (1977)
Comings, D.E., Okada, T.A.: Nuclear proteins. III. The fibrillar nature of the nuclear matrix. Exp. Cell Res. 103, 341–360 (1976)
Compton, J.L., Hancock, R., Oudet, P., Chambon, P.: Biochemical and electron microscopic evidence that the subunit structure of Chinese hamster ovary interphase chromatin is conserved in mitotic chromosomes. Europ. J. Biochem. 70, 555–568 (1976)
Cook, P.R., Brazell, I.A., Jost, E.: Characterization of nuclear structures containing superhelical DNA. J. Cell Sci. 22, 303–324 (1976)
Crossen, P.E., Pathak, S., Arrighi, F.E.: A high resolution study of the DNA replication patterns of Chinese hamster chromosomes using sister chromatid differential staining technique. Chromosoma (Berl.) 52, 339–347 (1975)
Hadlaczky, G., Sumner, A.T., Ross, A.: Protein-depleted chromosomes: I. Structure of isolated protein-depleted chromosomes. Chromosoma (Berl.) 81, 537–555 (1981a)
Hadlaczky, G., Sumner, A.T., Ross, A.: Protein-depleted chromosomes: II. Experiments concerning the reality of chromosome scaffolds. Chromosoma (Berl.) 81, 557–567 (1981b)
Hartwig, M.: Organization of mammalian chromosomal DNA: Supercoiled and folded circular DNA subunits from interphase cell nuclei. Acta Biol. Med. Germ. 37, 421–432 (1978)
Hittelman, W.N., Rao, P.N.: Mapping G1 phase by the structural morphology of prematurely condensed chromosomes. J. Cell Physiol. 95, 333–341 (1978)
Huberman, J.A., Riggs, A.D.: On the mechanism of DNA replication in mammalian chromosomes. J. molec. Biol. 32, 327–341 (1968)
Ide, T., Nakane, M., Anzai, K., Andoh, T.: Supercoiled DNA folded by non-histone proteins in cultured mammalian cells. Nature (Lond.) 258, 445–447 (1975)
Johnson, R.T., Rao, P.N.: Mammalian cell fusion: Induction of premature chromosome condensation in interphase nuclei. Nature (Lond.) 226, 717–722 (1970)
Lau, Y.-F., Arrighi, F.E.: Studies of mammalian chromosome replication: I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes. Cytogenet. Cell Genet. 27, 176–183 (1980)
Lau, Y.-F., Brown, R.L., Arrighi, F.E.: Induction of premature chromosome condensation in CHO cells fused with polyethylene glycol. Exp. Cell Res. 110, 57–61 (1977)
Lau, Y.-F., Hittelman, W.M., Arrighi, F.E.: Sister chromatid differential staining pattern in prematurely condensed chromosomes. Experientia (Basel) 32, 917–919 (1976)
Marsden, M.P.F., Laemmli, U.K.: Metaphase chromosome structure: evidence for a radial loop model. Cell 17, 849–858 (1979)
Meyn, R.E., Hewitt, R.R., Humphrey, R.M.: Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine. Exp. Cell Res. 82, 137–142 (1973)
Okada, T.A., Comings, D.E.: Higher order structure of chromosomes. Chromosoma (Berl.) 72, 1–14 (1979)
Okada, T.A., Comings, D.E.: A search for protein cores in chromosomes: Is the scaffold an artifact? Amer. J. Hum. Genet. 32, 814–832 (1980)
Pardoll, D.M., Vogelstein, B., Coffey, D.S.: A fixed site of DNA replication in eucaryotic cells. Cell 19, 527–536 (1980)
Paulson, J.R., Laemmli, U.K.: The structure of histone-depleted metaphase chromosomes. Cell 12, 817–828 (1977)
Rao, P.N., Johnson, R.T.: Induction of chromosome condensation in interphase cells. Advanc. Cell molec. Biol. 3, 135–189 (1974)
Rhome, D.: Prematurely condensed chromosomes of the Indian muntjac: A model system for the analysis of chromosome condensation and banding. Hereditas (Lund) 76, 251–258 (1974)
Sanbe, M., Aya, T., Ikeuchi, T., Sandberg, A.A.: Electron microscopic study of fused cells, with special reference to chromosome pulverization. J. nat. Canc. Inst. 44, 1079–1089 (1970)
Schwarzacher, H.G., Ruzicka, F., Sperling, K.: Electron microscopy of human banded and prematurely condensed chromosomes. Chromosomes today 5, 227–234 (1974)
Stubblefield, E.: Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence. Chromosome (Berl.) 53, 209–221 (1975)
Unakul, W., Johnson, R.T., Rao, P.N., Hsu, T.C.: Giemsa banding in prematurely condensed chromosomes obtained by cell fusion. Nature (New Biol.) (Lond.) 242, 106–107 (1973)
Vogelstein, B., Pardoll, M., Coffey, D.S.: Supercoiled loops and eucaryotic DNA replication. Cell 22, 79–85 (1980)
Wigler, M. Axel, R.: Nucleosomes in metaphase chromosomes. Nucleic Acids Res. 3, 1463–1471 (1976)
Worcel, A., Han, S., Wong, M.L.: Assembly of newly replicated chromatin. Cell 15, 969–977 (1978)
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Lau, Y.F., Arrighi, F.E. Studies of mammalian chromosome replication. Chromosoma 83, 721–741 (1981). https://doi.org/10.1007/BF00328530
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DOI: https://doi.org/10.1007/BF00328530