Incorporation of Tritium-Labeled Thymidine and Lysine Into Chromosomes of Cultured Human Leukocytes

Cave, M. Donald

doi:10.1083/jcb.29.2.209

Cited by 47 publications

(6 citation statements)

References 38 publications

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“…Examination of the data in many of these early publications (15)(16)(17)19) reveals that a small percentage of labeled metaphases was seen commonly after 1-to 2-h periods of labeling, a considerably shorter time than the 3-4 h estimated for G 2 . These data indicate that the length of G 2 in some cells may be markedly shorter than previously estimated.…”

Section: Discussionmentioning

confidence: 99%

Very late DNA replication in the human cell cycle

Widrow

Hansen

Kawame

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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G 2 was defined originally as the temporal gap between the termination of DNA replication and the beginning of mitosis. In human cells, the G 2 period was estimated to be 3-4 h. However, the absence of replicative DNA synthesis during this period designated G 2 has never been shown conclusively. In this report, we show that, at some autosomal and X linked loci, programmed DNA replication continues within 90 min of mitosis. Furthermore, the major accumulation of cyclin B1, a cell-cycle marker that is usually ascribed to G 2 , overlaps extensively with very late DNA replication. We conclude that the G 2 period is much shorter than previously thought and may, in some cells, be nonexistent.The eukaryotic cell cycle consists of an orderly series of events in which cells grow, replicate their DNA, and segregate the duplicated genome to the daughter cells. Our current textbook view of the cell cycle was first put forth by Howard and Pelc (1) as encompassing four phases: S phase, the time in which replicative DNA synthesis occurs; M phase, the time in which mitotic chromosome segregation and cell division proceed; and two gap phases, G 1 and G 2 , that temporally separate S phase from mitosis.The G 2 phase was inferred from the length of time between the addition of radiolabeled DNA precursors and the appearance of labeled, condensed mitotic chromosomes (1). Autoradiography, however, may not be sufficiently sensitive to detect low levels of replicative synthesis in the G 2 phase. The absence of detectable incorporation of radiolabel could thus be taken to indicate erroneously the absence of DNA replication. Furthermore, DNA repair synthesis can occur throughout the cell cycle (2, 3); replicative and repair DNA synthesis are not easily distinguishable by the autoradiographic method. An additional problem is that no cellular marker signaling the termination of replicative DNA synthesis has been identified. These features contribute to an inherent lack of both sensitivity and specificity for accurately defining the terminal border of S phase.Our studies on fragile sites and the replication timing of FMR1, the gene responsible for the fragile X syndrome, first brought our attention to the possibility of replication very close to mitosis (4). In cell lines derived from patients with fragile X syndrome, the replication of a large region surrounding the FMR1 gene is abnormally late, occurring within the G 2 ͞M compartment defined by DNA content (5, 6). Surprisingly, equally late replication was observed for several loci on the inactive X chromosome in normal female fibroblasts (7,8), as well as on the inactive X chromosome in human-hamster hybrids (8). In this report, we have determined more precisely the timing of late DNA replication in the human cell cycle, by using a new, sensitive method for fractionating cells late in the cell cycle (9). We address two questions. Does this very late replication occur during what is usually termed G 2 ? What proportion of the genome replicates in this late compartment? MATERIALS A...

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Section: Discussionmentioning

confidence: 99%

Very late DNA replication in the human cell cycle

Widrow

Hansen

Kawame

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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“…These results are at variance with those of Prescott and Bender (1963) and of Prensky and Smith (1964) but may reflect differences, in these reports, in the proteins retained by the chromosomes. Cave (1966) and Zweidler (1965) both find conservation of chromosomal protein.…”

Section: ~mentioning

confidence: 98%

“…Unfortunately, these conditions are not met. Such methods as treatment with hypotonic solutions and acetic acid or acetic alcohol, which permit isolation of the spindle and metaphase chromosomes (Prescott and Bender, 1961) and which effect sufficient softening to permit the high degree of flattening convenient for radioautographic study of chromosomes, also result in the removal of some or most histones (Cave, 1966). The basic approach, as described above, was used with modifications in an attempt to detect the synthesis of histone-like chromosomal proteins in the midst of other proteins.…”

Section: Introductionmentioning

confidence: 99%

The Syntheses of Deoxyribonucleic Acid and Histone in the Onion Root Meristem

Bloch

MacQuigg

Brack

et al. 1967

The Journal of Cell Biology

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A comparison of the times necessary to incorporate tritium-labeled lysine and arginine into histones and tritium-labeled thymidine into DNA indicates that the periods of DNA and histone synthesis prior to division closely coincide. (The comparison was made by determining the times necessary, after pulse labeling, for cells with marked chromosomes to enter and then leave the division stages.) An additional period of chromosomal protein synthesis, of short duration, occurs late in interphase. Most of the chromosomal proteins appear either to be synthesized in the nucleus or to migrate there shortly after synthesis. Much of this protein is conserved from one division to the next. Studies of the effects of puromycin and fluorodeoxyuridine on the syntheses of DNA and histone suggest that continuation of DNA synthesis is dependent on a concurrent protein synthesis. Histone synthesis, on the other hand, can proceed at a normal rate under conditions in which DNA synthesis is inhibited.

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“…However, to our knowledge only a few studies (Cave, 1966;Cave, 1967) have dealt with the time sequence o f protein synthesis in vertebrate sex chromosomes, as compared to that o f DNA synthesis. Cave (1967) reports that in contrast to the distinct patterns o f labeling by 3H-thymidine, especially with regard to one of the X chromosomes in the human female, none o f the human chromosomes is distinctively asynchronous in 3H-lysine incorporation.…”

Section: Introductionmentioning

confidence: 99%

The pattern of protein synthesis in late S and G<sub>2</sub> of bovine sex chromosomes

Herzog¹,

Steffensen²

1968

Cytogenet Genome Res

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The temporal relation and correlation between the syntheses of DNA and protein of bovine female sex chromosomes were studied. Significant differences were found between the patterns of protein and DNA synthesis as studied by ³H-lysine and ³H-arginine incorporation into protein and ³H-thymidine incorporation into DNA. In contrast to the late-labeling X chromosome for DNA, no X chromosome was found to be late labeling with respect to protein containing either ³H-lysine or ³H-arginine. No distinct differences were found between the patterns of incorporation of the two tritiated amino acids into the chromosome protein.

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Incorporation of Tritium-Labeled Thymidine and Lysine Into Chromosomes of Cultured Human Leukocytes

Cited by 47 publications

References 38 publications

Very late DNA replication in the human cell cycle

Very late DNA replication in the human cell cycle

The Syntheses of Deoxyribonucleic Acid and Histone in the Onion Root Meristem

The pattern of protein synthesis in late S and G<sub>2</sub> of bovine sex chromosomes

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