How does inactivation change timing of replication in the human X chromosome?

Summary The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G₁/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuos labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.     

Chronology and pattern of replication in the bone marrow chromosomes of Gallus domesticus

The duration of the cell generation, the chronology, and the pattern of chromosome duplication was studied in the bone marrow of Gallus domesticus. The duration of the phases of the cell cycle is: cell generation 17.5 hours, S period 9 hours. G₂ period plus prophase stage 2.5 hours, G₁ period 6 hours. Chromosome replication begins at many sites. During middle S it extends to the whole complement and finally finishes in small, late replicating regions of the macrochromosomes. Interchromosomal asynchrony of duplication at the initiation or at the end of the S period was not observed. Z-chromosomes begin and finish DNA synthesis synchronously with the other macrochromosomes. The W-chromosome in females is the last microchromosome to finish replication. However it ends DNA synthesis at about the same time as the macrochromosomes. Similarities and differences between chromosome replication in Aves and Mammalia are considered.     

Replication and G2 checkpoints: their response to caffeine

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Bi-parametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G₂ checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G₂ checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G₂ block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine. Received: 21 February 2000 / Accepted: 31 July 2000     

Pattern of chromosomal replication in synchronized lymphocytes

Summary The BrdUrd-Hoechst 33258-Giemsa technique was employed to study patterns of chromosomal replication in human lymphocytes synchronized by Methotrexate (MTX). It is proposed that in the presence of MTX, a major portion of the cell population is blocked in an advanced stage of the S-phase and not in the G₁/S border of the cell cycle. At this point, the replication of the chromosomal segments corresponding to the R-bands is terminated, and the replication of the G-bands and the inactive X-chromosomes is being initiated. The use of this method in the study of higher resolution patterns of chromosomal replications is proposed.     

DNA REPLICATION PATTERNS AND CHROMOSOMAL PROTEIN SYNTHESIS IN OPOSSUM LYMPHOCYTES IN VITRO

DNA replication patterns were determined in the autosomes and sex chromosomes of phytohemagglutinin-stimulated lymphocytes from the opossum (Didelphis virginiana) by employing thymidine-³H labeling and high-resolution radioautography. Opossum chromosomes are desirable experimental material due to their large size, low number (2n = 22), and morphologically distinct sex chromosomes. The autosomes in both sexes began DNA synthesis synchronously and terminated replication asynchronously. One female X chromosome synthesized DNA throughout most of the S phase. Its homologue, however, began replication approximately 3.5 hr later. The two X's terminated DNA synthesis synchronously, slightly later than the autosomes. This form of late replication, in which one X chromosome begins DNA synthesis later than its homologue but completes replication at the same time as its homologue, is apparently unique in the opossum. The male X synthesized DNA throughout S while the Y chromosome exhibited late-replicating characteristics. The two sex chromosomes completed synthesis synchronously, slightly later than the autosomes. Grain counts were performed on all chromosomes to analyze trends in labeling intensity at hourly intervals of S. By analyzing the percent of labeled mitotic figures on radioautographs at various intervals after introduction of arginine-³H, chromosomal protein synthesis was found not to be restricted to any portion of interphase but to increase throughout S and into G₂.     

Analysis of chromosome replication by a BrdU antibody technique

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 (S_E and S_L) 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 S_E or S_L. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.     

DNA replication sequence of human chromosomes in blood cultures

Summary The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G ₂ periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H³. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S₁ to S₄.The first chromosomes to replicate were Nos. 1, 3, 5 and X followed by the Nos. 2, 4 and several chromosomes of groups 6–12, 13–15 and 19–20. Later the pairs 16, 17, 18 and the chromosomes of group 21–22 replicated. Chromosome Y in the male was the last to replicate, beginning its duplication when all the other chromosomes had reached the intermediate S stage.The earliest chromosomes to finish the duplication were Nos. 19, 20 and 21 followed by Nos. 16, 17, 18, 22 and the chromosomes of group 13–15. Afterward and at about the same time the replication of pairs 2, 4, 6, 8, the X and Y chromosomes in the male and one X chromosome in the female concluded. The other X chromosome in the female was the last to end its duplication appearing totally labelled until the final stage of the S period.Replication of the long and medium size chromosomes begins at localised regions, then extends over the total length of the chromosome and at the end of the S stage takes place only in small zones different from those replicating early.Asynchrony between homologous chromosomes was observed at the beginning and at the end of the S period.     

Location of the mouse complement factor H gene (cfh) by FISH analysis and replication R-banding.

A technique for replication R- and G-banding of mouse lymphocyte chromosomes was developed, and the replication R-banding pattern was analyzed. Optimal banding patterns were obtained with thymidine- and BrdU-treatment of lymphocytes in the same cell cycle. This produced replication R-band patterns that were the complete reverse of the G-band patterns on all chromosomes. Replication R-banding methods can be used in conjunction with nonisotopic, fluorescence in situ hybridization (FISH) to localize cloned probes to specific chromosomal bands on mouse chromosomes. with these methods the mouse complement factor H gene (cfh) was localized to the terminal portion of the F region of Chromosome 1. Q-banding patterns were also obtained by the replication R-banding method and may be useful for rapid identification of each chromosome.     

Cytogenetic replication studies with short thymidine pulses in bromodeoxyuridine-substituted chromosomes of different mouse cell lines

Summary In normal diploid fibroblasts of the mouse, 3T3-, SV-3T3-, and Meth A-cells, the chromosome replication patterns were studied by a bromodeoxyuridine (BrdU)-labelling technique. SV-3T3 is a subline of 3T3 transformed by SV 40 and Meth A is a permanent cell line from Balb c transformed by methylcholanthrene. The use of 1 h thymidine pulses permits high resolution of the S-phase after partial synchronization of the cells at G₁/S in an otherwise BrdU-substituted S-phase. It could be shown that the autosomal heterochromatin of the mouse (Mus musculus) starts replication during the early S-phase (R-band replication), continues while R-band chromatin finishes, and still replicates when G-band chromatin starts. The heterochromatin finishes before the majority of G-bands have been replicated. There is no fundamental difference in the course of chromosome replication between the different cell lines studied here. It is concluded that there are no obligate changes in the course of the S-phase linked to the process of transformation.     

Studies of mammalian chromosome replication

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 m 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.     

ACTIVITY OF TUBULOSINE ON THE KINETICS OF ROOT MERISTEM CELL POPULATION

The action of tubulosine on the mitotic cycle was studied using continuous labelling with tritiated thymidine. This alkaloid provokes a lengthening of the G₁ and S phases and a blocking of G₂ is totally reversible when the treatment is followed by recovery in normal medium. At a dose of tubulosine which induces a reversible mitostasis in the shortest possible time the lengthening of the phases of the cell cycle was estimated by three different techniques: labelled mitoses for the determination of G₂; labelling intensity for the determination of S; binucleate cells for the determination of T, and an original technique using labelling index of binucleate cells for the determination of G₁. The limits of the technique of labelled mitosis together with the interest of the technique aiming at the direct determination of G₁ in the case of a perturbed cycle are then discussed.     

Alterations in the time of X chromosome replication induced by 5-azacytidine in a patient with 48,XXXY/47,XXY

It has recently been shown that 5-azacytidine (5-azaC) can induce altered replication patterns of the late-replicating X chromosome in normal female cells. This has been demonstrated by bromodeoxyuridine labelling of cells late in the S phase. In the present study the same method was applied to the lymphocytes of a Klinefelter patient (48,XXXY/47,XXY). Significant 5-azaC-induced changes in the replication of the entire inactive X chromosome, from late to early, were found in the lymphocytes of this patient. These results indicate that hypomethylating agents can not only alter the replication of individual bands, but also change the gross replication schedule of multiple inactive X chromosomes in the presence of a Y chromosome.     

Cell-specific variation of X chromosome replication in the Syrian hamster (Mesocricetus auratus)

The sub-division of S-phase in Syrian hamsters, on the basis of BrdU/Hoechst 33258/Giemsa banding, has allowed a quantitative comparison of the replication of individual chromosome bands within defined subphases of S. This analysis has shown that in hamsters, as has been reported in humans, there are distinct patterns of early replication in vitro in the early X, the late X in fibroblasts, and the late X in lymphocytes. In addition, it has been possible to show that, although the pattern of replication of the late X in fibroblasts differs from that in lymphocytes, the time in S at which bands first appear on this chromosome is the same in the two cell types. — No significant heterogeneity can be ascribed to differences between individuals, adult or embryonic sources, culture media, or time of exposure to BrdU. — The absence of any detectable heterogeneity in the replication band frequencies in autosomal heterochromatic arms suggests that the cell-specific variability of the late-replicating X is a feature of facultative rather than constitutive heterochromatin.     

A Rapid Automated Stathmokinetic Method For Determination of In Vitro Cell Cycle Transit Times

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G₁. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G₂+ M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.     

STUDIES ON LYMPHOCYTES

Some of the time parameters of the cell cycle in bovine thoracic duct lymphocytes have been estimated by analysing labeled mitoses curves, and by double labeling. The two methods gave similar estimates of T_s. Thus, T_s measured directly from labeled mitoses curves varied from 4 to 6 hr, while the estimates from double labeling were 4.8 and 4.5 hr. T _G measured directly from labeled mitoses curves was 5 hr, and estimates of T_G from the values of T_s ranged from 6.3 to 7.7 hr. The present data confirm the short generative cycle of large thoracic duct lymphocytes. Extracorporeal irradiation of the lymph (ECIL) had no detectable effect on the cell cycle or the fractional production rate of the lymphocytes. However, the calculated absolute production was reduced following ECIL, due to a decrease in the absolute number of cells present. The grain count over mitoses after ECIL was approximately one-half that found before ECIL.     

Altered cell cycle progression and aberrant mitosis in adenovirus-infected rodent cells

Actively growing mouse or rat embryo cells suffered structural chromosome damage, mitotic anomalies, and polyploidy after infection by human adenovirus type 5. Chromosome damage required expression of one or more early viral genes and showed regular periodicity in its frequency. The growth cycle time of some of the infected cells was reduced by about 5 hours due to a decrease in G₁, and the interval between successive waves of chromosome damage corresponded to this reduced cycle time. After infection there was a decrease in cells with G₁ DNA content and an increase in cells with G₂ diploid, aneuploid, and polyploid DNA contents. We suggest these effects are due to the expression in semipermissive cells of early viral gene(s), whose function in productive infection in vivo is to alter cell cycle controls in order to maximize the number of cells able to replicate viral DNA and the time such cells spend in DNA replication.     

Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

Mammalian cell fusion

The behaviour of heterochromatin during premature chromosome condensation (PCC) was studied in a cell line of Microtus agrestis after fusion with mitotic HeLa cells. In the G₁- and G₂-PCC, the heterochromatic nature of the X-chromosomes was detectable by their intense staining. The pulverized appearance of the S-phase PCC was correlated with incorporation of ³H TdR into the DNA. Three types of S-PCC were observed. PCC with a pulverized appearance of: (a) only the autosomes (early S); (b) autosomes and X-chromosomes (mid S); and (c) only the X-chromosomes (late S). The behaviour of heterochromatin during replication, as observed by the PCC method, was no different from that of euchromatin. The data on the sequence of chromosome replication indicate that the centromeric regions of the X-chromosomes were the last segments to replicate. The completion of DNA synthesis in the X-chromosomes appears to be followed by progressive chromosome condensation during G₂ even before the actual initiation of prophase.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase