BrdU-Hoechst-Giemsa analysis of DNA replication in synchronized lymphocyte cultures

The combination of a technique of reversible methotrexate (MTX) imposed G₁/S block in cultures of human lymphocytes with the BrdU-Hoechst-Giemsa technique permitted the study of DNA replication patterns in individual chromosomes at different intervals of the S phase in a cell cohort with uniform S + G₂ duration. The procedure did not increase either the frequency of chromosomal breakage or SCE freqeuncy. The technique applied permitted visualization of the banding pattern in over 90% of mitoses. Examination of mitoses following different times of exposure to BrdU revealed a high degree of synchrony in the progression of the cell cohort examined through the S phase.The presence of two distinct late replication patterns of the allocyclic X chromosome was confirmed in studies on lymphocytes from normal human females by this technique. Interindividual and intercellular differences of the replication pattern have been demonstrated. The replicating patterns from one individual were relatively constant.The analysis of the Y chromosome has revealed marked differences of the termination of replication in individual cells. Euchromatic regions have been shown to complete DNA synthesis first, followed by the distal part of the long arm and, finally, by the region of Yq11/Yq12 junction. Lateral asymmetry was localised at this region.This paper was presented as a preliminary communication at the Helsinki Chromosome Conference Aug. 29–31, 1977, and, in its final form, at the 7th International Chromosome Conference, Oxford, Aug. 26–30, 1980.     

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.     

Patterns of chromosomal fragmentation due to uracil-DNA incorporation reveal a novel mechanism of replication-dependent double-stranded breaks

There is growing evidence that spontaneous chromosomal fragmentation, one of the main contributors to genetic instability, is intimately linked to DNA replication. In particular, we proposed before that uracil incorporation in DNA triggers chromosomal fragmentation due to replication fork collapse at uracil-excision intermediates. We tested predictions of this model at the chromosomal level in the dut mutants of Escherichia coli , by determining the relationship between DNA replication and patterns of fragmentation in defined chromosomal segments. Here we show that the uracil-DNA-triggered chromosomal fragmentation: (i) has a gradient that parallels the replication gradient, (ii) shows polarity within defined segments pointing towards replication origins and (iii) reorganizes to match induced replication gradients, confirming its dynamic pattern. Unexpectedly, these fragmentation patterns not only support the replication fork collapse model, but also reveal another mechanism of the replication-dependent chromosomal fragmentation triggered by uracil excision.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

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.     

The Two Cis-Acting Sites,parS1 and oriC1, Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (ori_I) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the ori_I region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and ori_I polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Author summary

Proper chromosome organisation within the cell is crucial for cellular proliferation. However, the mechanisms driving bacterial chromosome segregation are still strongly debated, partly due to their redundancy. Two patterns of chromosomal organisation can be distinguished in bacteria: a transversal chromosomal arrangement, such as in E. coli, where the origin of replication (ori) is positioned at midcell and flanked by the two halves of the chromosome (replichores), and a longitudinal arrangement, such as in C. crescentus, where ori is recruited to the pole and the replichores extend side by side along the long axis of the cell. Here, we present the first detailed characterization of the arrangement of the genetic material in a multipartite genome bacterium. To this end, we visualised the position of 19 loci scattered along the two V. cholerae chromosomes. We demonstrate that the two chromosomes, which both harbour a Par system, are longitudinally organised. However, the smaller one only extended over the younger cell half. In addition, we found that disruption of the Par system of chromosome I released its origin from the pole but preserved its longitudinal arrangement. Finally, we show that the addition of an ectopic ori perturbed this arrangement, suggesting that the replication program contributes to chromosomal organisation.     

Replication control of autonomously replicating human sequences.

Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system.     

RPA is an initiation factor for human chromosomal DNA replication

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.     

Computer image analysis of variance between human chromosome replication sequences and G-bands.

A computer image analysis system was applied to the quantitative study of chromosomal early- and late-replication patterns from the leukocytes of several normal human donors, and these patterns were compared with the chromosomal G-banding patterns. The first and last few hours of replication were discriminated by selective bromodeoxyuridine vs. thymidine incorporation in DNA and a Hoechst-blacklight-Giemsa stain technique. Image analysis with Tufts Piquant system involved automatic determination of chromosome boundaries, centromeres and telomeres, linear chromatid axes, chromatid density measurements along each axis, and comparative length normalized density profiles for each chromatid and the chromosome. Consistent complementary early- and late-replication patterns were determined for autosomes 1-6 and the X chromosomes. Limited intracellular or interindividual variability occurred in the intensity of a few active replication peaks but not in their location. However, there were very distinct regions of noncorrespondence between the late-replication patterns and the G-band patterns, in contrast with previous observations, although many similarities were also evident. These differences are interpreted with reference to a general model of replication sequence control of cell differentiation.     

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH₂-10-CH₃PteGlu₄). When the time-dependent synthesis of MTX polyglutamates (4-NH₂-10-CH₃PteGlu₂ and higher) at extracellular concentrations of 10 and 100 μm methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μm is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μm intracellular methotrexate, respectively, after 24 h in the presence of 100 μm extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μm with both cell types which corresponded to 10 to 12 μm intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu_4–5) are more avidly retained by the cells than the shorter ones (Glu_2–3).     

Spatio-temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cells

The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains.     

Yeast Two-Hybrid Analysis of PriB-Interacting Proteins in Replication Restart Primosome: A Proposed PriB–SSB Interaction Model

PriB is one of the components of the replication restart primosome. The activity mediated by the primosomal proteins, including PriB, is required for reinitiating chromosomal DNA replication in bacteria after DNA damage. As such, the study of the interactions between PriB and members in the primosome is essential to better understand their mechanism of action. In this study, we investigated PriB-interacting proteins in the primosome through the yeast two-hybrid system. Yeast two-hybrid analysis using two strains, Y187 and AH109, revealed that PriB interacts with PriA, DnaT, SSB, and itself and does not interact with DnaA, DnaB, DnaC, or DnaG. In addition, mutational analysis showed that PriB may bind to SSB via K82, K84, and K89 in the L₄₅ loop. Based on these preliminary data, we proposed a PriB–SSB interaction model. Further work can focus on determination of how PriB binds to the PriA–SSB complex in replication restart.     

Cytoplasmic alteration of an established pattern of Rana pipiens chromosomal DNA replication

Cells from Rana pipiens embryos were incubated in ³H-thymidine for the duration of the last quarter of the S period plus the G₂ period of the cell cycle. Chromosomes of animal hemisphere cells of stage 9 embryos showed uniform labeling, whereas chromosomes of endodermal cells of stage 17 embryos showed terminal labeling. We tested whether egg cytoplasm would alter an established temporal pattern of chromosomal DNA replication. Nuclei from disaggregated endodermal cells of stage 17 embryos were transplanted into activated and enucleated eggs. The eggs were then allowed to develop to the blastula stage. Animal hemisphere explants of these blastulae were incubated in ³H-thymidine. Radioautographic localization of silver halide grains demonstrated a chromosomal DNA replication pattern that was uniform over the the metaphase chromosomes. The egg cytoplasm had evidently altered an established temporal pattern of chromosomal DNA replication.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

The influence of carbohydrate ligands on the cytotoxicity of liposomes bearing a methotrexate-diglyceride conjugate in human acute leukemia cell cultures

The efficiency of the chemotherapeutic agent methotrexate (MTX) in cancer treatment is limited by the frequent development of the drug resistance of tumor cells. We had previously shown in vitro using human acute leukemia cells with various sensitivity to MTX (T-lymphoblastic CCRF-CEM line and resistant CEM/MTX subline) that MTX incorporation into liposomes in the form of a lipophilic prodrug, diglyceride conjugate (MTX-DG), allows for the overcoming of cell resistance due to the impaired active transmembrane transport. In this work, we have studied the profile of binding with carbohydrates of the cell lines mentioned using carbohydrate fluorescent probes (poly(acryl amide) conjugates). Lipophilic conjugates of tetrasaccharide SiaLe^X, 6′-HSO₃LacNAc, and also inactive pentaol for incorporation into liposomes, have been synthesized. The cytotoxicity of MTX-DG liposomes equipped with the SiaLe_X ligand toward the sensitive CCRF-CEM cell culture was demonstrated to be 3.5 times higher than that of MTX-DG liposomes bearing the control inactive pentaol. The activity of MTX liposomes bearing 6′-HSO₃LacNAc toward resistant CEM/MTX was 1.6-fold increased. The use of carbohydrate ligands as molecular addresses for drug-carrying liposomes as a potential method of treating heterogeneous tumor tissue is discussed.     

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.     

The majority of G0 transgenic mice are derived from mosaic embryos

Most transgenic mice are generated by the direct microinjection of DNA fragments into the pronuclei of fertilized eggs. It has been generally assumed that the majority of integration events occur prior to the first round of chromosomal DNA replication (Palmiter and Brinster, 1986). In this study we have determined by comparison of PCR, Southern blot and transmission frequencies that at least 62% of integration events generate a mosaic (somatic and/or germline) G₀ transgenic mouse. Furthermore, the statistical probability of transgene-containing cells segregating to the various early embryo lineages implies that this is probably an underestimate of the true mosaic frequency. Thus, the majority of DNA injected into fertilized mouse eggs integrates after the first round of chromosomal DNA replication, therefore most G₀ transgenic mice are derived from a mosaic embryo.     

DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI

It is generally believed that DNA replication in most eukaryotes proceeds according to a precise program in which there is a defined temporal order by which each chromosomal region is duplicated. However, the regularity of this program at the level of individual chromosomes, in terms of both the relative timing and the size of the DNA domain, has not been addressed. Here, the replication of chromosome VI from synchronized budding yeast was studied at a resolution of ∼ 1 kb with DNA combing and fluorescence microscopy. Contrary to what would be expected from cells following a rigorous temporal program, no two molecules exhibited the same replication pattern. Moreover, a direct evaluation of the extent to which the replication of distant chromosomal segments was coordinated indicates that the overwhelming majority of these segments were replicated independently. Importantly, averaging the patterns of all the fibers examined recapitulates the ensemble-averaged patterns obtained from population studies of the replication of chromosome VI. Thus, rather than an absolutely defined temporal order of replication, replication timing appears to be essentially probabilistic within individual cells, exhibiting only temporal tendencies within extended domains.     

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.