Abstract
Chromosome replication was studied without synchronization in human lymphocyte and amniotic cell cultures visualizing very short 5-bromodeoxyuridine (BrdU) pulses by an immunologic technique (BAT). The findings agree in general with those facts known from earlier BrdU staining techniques. The very high sensitivity of BAT was shown to allow the detection of replication in a band where 1 in 200 nucleotides is replaced by BrdU. The main observations are: though the replication patterns after BAT appear strange the bands correspond to those described by the Paris Conference (1971). At the beginning of the S-phase a stepwise onset of replication in only a subset of R-bands is confirmed. There is a considerable difference in the sensitivity between early and late S (SE and SL) for the detection of BrdU pulses. This difference probably reflects a different spatial arrangement of chromatin in R-bands as compared with G-bands below the level of cytogenetic analysis. The use of short pulses did not reveal any additional subdivision of SE or SL. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.
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References
Adolph S (1988) In situ nick translation distinguishes between C- band positive regions on mouse chromosomes. Chromosoma 96:102–106
Begg AC, McNally NJ, Shrieve DC, Kärcher H (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6:620–626
Calderon D, Schnedl W (1973) A comparison between quinacrine fluorescence banding and 3H-thymidine incorporation. Humangenetik 18:63–70
Calza RE, Eckhardt L, DelGiudice T, Schildkraut CL (1984) Changes in gene position are accompanied by a change in time of replication. Cell 36:689–696
Camargo M, Cervenka J (1982) Patterns of DNA replication of human chromosomes. II. Replication map and replication model. Am J Hum Genet 34:757–78
Dolbeare F, Gratzner H, Pallavicini MG, Gray JW (1983) Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Cell Biol 80:5573–5577
Dutrillaux B (1975) Traitements discontinus par le BrdU et coloration par l'acridine orange: obtention de marquages R, Q et intermediaires. Chromosoma 52:261–273
Dutrillaux B, Laurant C, Couturier J, Lejeune J (1973) Coloration par l'acridine orange de chromosomes préalablement traités par le 5-bromodeoxyuridine (BUDR). C R Acad Sci 276:3179–3181
Dutrillaux B, Couturier CL, Viegas Pegnignont E (1976) Sequence of DNA replication in 227 R and Q-bands of human chromosomes using a BrdU treatment. Chromosoma 58:51–61
Fox MH (1980) A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry. Cytometry 1:71–77
Ganner E, Evans HJ (1971) The relationship between pattern of DNA replication and of quinacrine fluorescence in the human chromosome complement, Chromosoma 35:326–341
Gjerset RA, McCarthey BJ (1977) Limited accessibility of chromatin satellite DNA to RNA polymerase from E. coli. Proc Natl Acad Sci USA 74:4337–4340
Hand R (1978) Eucaryotic DNA: Organization of the genome for replication. Cell 15:317–325
Holmquist GP (1987) Role of replication time in the control of tissue-specific gene expression. Am J Hum Genet 40:151–173
Kaelbling M, Miller DA, Miller DJ (1984) Restriction enzyme banding of mouse metaphase chromosomes. Chromosoma 90:128–132
Kapp LN, Painter RB (1982) X ray inhibition of DNA synthesis at discrete times during S phase in synchronous human diploid fibroblasts. Radiat Res 89:424–427
Karube T, Watanabe S (1988) Analysis of the chromosomal DNA replication pattern using the bromodeoxyuridine labelling method. Cancer Res 48:219–222
Kim MA, Johannsmann R, Grzeschik K-H (1975) Giemsa staining of the sites replicating DNA early in human lymphocyte chromosomes. Cytogenet Cell Genet 15:363–371
Latt SA (1973) Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395–3399
Lau YF, Arrighi E (1981) Studies of mammalian chromosome replication. II. Evidence for the existence of defined chromosome replicating units. Chromosoma 83:721–741
Paris Conference (1971) Standardization in human cytogenetics. Birth defects: Original article series, VIII: 7,1972. The National Foundation, New York
Reddy KS, Savage JRK, Papworth DG (1988) Replication kinetics of X chromosomes in fibroblasts and lymphocytes. Hum Genet 79:44–48
Richter A, Hand R (1979) DNA replication during a serum-induced S-phase in primate CV — 1 cells. Exp Cell Res 121:363–371
Savage JRK, Bhunya SP (1980) Cytological sub-division of S-phase in the Syrian hamster (Mesocricetus auratus). Chromosoma 77:169–180
Savage JRK, Prasad R (1984) Cytological subdivision of S-phase of human cells in asychronous culture. J Med Genet 21:204–212
Savage JRK, Prasad R, Papworth DG (1984) Subdivision of S phase and its use for comparative purposes in cultured human cells. J Theor Biol 111:355–367
Schempp W, Vogel W (1978) Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU. Chromosoma 67:193–199
Schmid (1963) DNA replication patterns of human chromosomes. Cytogenetics 2:175–193
Schmidt M (1980) Two phases of DNA replication in human cells. Chromosoma 75:101–110
Schwarzacher HG, Wolf U (1974) Methods in human cytogenetics. Springer, Berlin, Heidelberg, New York
Schwemmle S, Mehnert K, Vogel W (1989) How does inactivation change timing of replication in the human X-chromosome. Hum Genet 83:26–32
Sheck LE, Muirhead KA, Horan PK (1980) Evaluation of the S phase distribution of flow cytometric DNA histograms by autoradiography and computer algorithms. Cytometry 1:109–117
Speit G (1984) Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes. Hum Genet 67:264–269
Speit G, Haupter S (1985) On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes. II. Differences between the demonstration of sister chromatid differentiation and replication patterns. Hum Genet 70:126–129
Speit G, Vogel W (1986) Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody. II. Demonstration of sister chromatid exchanges. Chromosoma 94:103–106
Taylor JH (1960) Asynchronous duplication of chromosomes in cultured cells of chinese hamster. J Biophys Biochem Cytol 7:455–463
Taylor JH, Woods PS, Hughes WL (1957) The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labelled thymidine. Proc Natl Acad Sci USA 43:122–128
Vogel W, Reisacher A (1987) The proportion of G- and R-band chromatin determined by comparison of replication patterns and FCM-analysis. In: Burger C, Ploem JS, Goertter K (eds) Clinical cytometry. Academic Press, London
Vogel W, Speit G (1986) Cytogenetic replication studies with short thymidine pulses in bromodeoxyuridine-substituted chromosomes of different mouse cell lines. Hunt Genet 72:63–67
Vogel W, Schempp W, Sigwarth I (1978) Comparison of thymidine, fluorodeoxyuridine, hydroxyurea and methotrexate blocking at the G/S-phase transition of cell cycle studied by replication patterns. Hum Genet 45:193–198
Vogel W, Autenrieth M, Speit G (1986) Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine-antibody. Hum Genet 72:129–132
Yurov YB (1980) Rate of DNA replication fork movement within a single mammalian cell. J Mol Biol 136:339–342
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Vogel, W., Autenrieth, M. & Mehnert, K. Analysis of chromosome replication by a BrdU antibody technique. Chromosoma 98, 335–341 (1989). https://doi.org/10.1007/BF00292386
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DOI: https://doi.org/10.1007/BF00292386