Summary
In vivo exposure to 5-azacytidine (10−6M) depressed the incorporation of methyl groups to GC rich regions ofAllium cepa L. DNA. Nearly 22% of its 5-methylcytosine residues were under-methylated. The treatment stimulated 1.8 times the rate of [3H]uridine incorporation, as measured in meristems proliferating under steady state kinetics. Nucleologenesis was shortened from 2.7 to 1.6 h in synchronous binucleate cells after 5-azacytidine treatment lasting the whole S period of their previous interphase. By hypomethylating DNA sequences replicated at different times during the S period, it could be inferred that the cistron replication took place in early S. Thus, nucleogenesis was shortened to only 0.6 h after such treatment. Sequential short treatment periods using [3H]thymidine confirmed that the nucleolar organizer regions of the chromosomes replicate in early S.
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Abbreviations
- A:
-
adenine
- AZA:
-
5-azacytidine
- C:
-
cytosine
- EDTA:
-
ethylenediamine tetraacetic acid
- mC:
-
5-methylcytosine
- T:
-
thymine
- TE:
-
tris-HCl EDTA buffer
- HPLC:
-
high pressure liquid chromatography
- NOR:
-
nucleolar organizer region
- PPO:
-
2,5-diphenyloxazole
- POPOP:
-
1,4-di[2(5-phenyloxazolyl) benzene
- TCA:
-
trichloroacetic acid
- tris:
-
tris (hydroxy-methyl) aminomethane
References
Balazs I, Schildkraut CL (1971) DNA replication in synchronized cultured mammalian cells. II. Replication of ribosomal cistrons in thymidine-synthesized Hela cells. J Mol Biol 57: 153–158
Benavente R, Rose KM, Reimer G, Hügle-Dörr B, Scheer U (1987) Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells. J Cell Biol 105: 1483–1491
Bird AP (1984) DNA methylation versus gene expression. J Embryol Exp Morphol 83 [Suppl]: 31–40
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254
Boyes J, Bird AP (1991) DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 64: 1123–1134
Cabello P, González-Fernández A, Leyton C, Sans J, De la Torre C (1986) Stimulated nucleologenesis in meristematic cells with hypomethylated DNA. Biol Cell 58: 91–94
Cedar H (1988) DNA methylation and gene activity (mini review). Cell 53: 3–4
De la Torre C, Fernández-Gómez ME, Giménez Martín G (1975) Rate of nucleologenesis as a measure of gene activity. Nature 256: 503–505
Fernández-Gómez ME, Stockert JC, López-Sáez JF, Giménez Martín G (1969) Staining plant cell nucleoli with AgNO3 after formalin-hydroquinone fixation. Stain Technol 44: 48–49
Flavell RB, O'Dell M, Thompson WF (1988) Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat. J Mol Biol 204: 523–534
Giacomoni D, Finkel D (1972) Time of duplication of ribosomal RNA cistrons in a cell line ofPotorus tridactylis (rat kangaroo). J Mol Biol 70: 725–728
Giménez-Martín G, González-Fernández A, López-Séz JF (1965) A new method of labelling cells. J Cell Biol 26: 305–109
—, De la Torre C, Fernández-Gómez ME, González-Fernández A (1974) Experimental analysis of nucleolar reorganization. J Cell Biol 60: 502–507
Morcillo G, De la Torre C (1979) Mapping nucleologenesis in relation to transcription. Biol Cell 36: 1–6
Ochs RL, Liwsche MA, Shen E, Carrol RE, Busch H (1985) Nucleologenesis: composition and fate of prenucleolar bodies. Chromosoma 92: 330–336
Scheer V, Benavente R (1990) Functional and dynamic aspects of mammalian nucleolus (review article). BioEssays 12: 14–21
Stambrook PJ (1974) The temporal replication of ribosomal genes in synchronized Chinese hamster cells. J Mol Biol 82: 303–313
Vanyushin BF (1984) Replicative DNA methylation in animals and higher plants. Curr Top Microbiol Immunol 108: 99–112
Watson JC, Kaufman LS, Thompson WF (1987) Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes ofPisum sativum. J Mol Biol 193: 15–26
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Mergudich, D., Leyton, C., Gonzalez-Fernandez, A. et al. Determination of the replication time of nucleolar organizer DNA after 5-azacytidine treatment for restricted parts of the S period. Protoplasma 167, 43–48 (1992). https://doi.org/10.1007/BF01353579
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DOI: https://doi.org/10.1007/BF01353579