Incorporation of proteins labeled with H3-lysine into chromosomes of human leucocytes cultured in vitro

The experiments were carried out on human leucocytes cultured in vitro. We studied the distribution of silver grains over metaphase chromosomes after pulse-labeling of cells with H³-lysine in S- and G2 phases. It was found that the grain number per chromosome of the pairs No. 1–3, 13–15 is proportional to their lengths. The probability of incorporation of labeled proteins into each of the homologous chromosomes of the first pair is equal to 0.5 found from the results of statistical analysis of silver grain counts. In cells with karyotype XXX labeled in late-S, the grain number per chromosome in the subgroup 6-X-7 is uniform. In these cells there is no difference in labeling densities among chromosomes of the group A. The data obtained suggest that the formation of the protein component in autosomes of different groups as well as in homologous autosomes and sex-chromosomes proceeds simultaneously and at equal rate.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

Chronology of chromosome reduplication in Chinese hamster cells incubated at low temperature

Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H³-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H³-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment.     

Chromosome banding and DNA replication patterns in bird karyotypes

The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence.     

Different DNA replication behavior of a tandem duplication in Drosophila melanogaster

Salivary gland chromosome DNA replication of a heterozygous tandem duplication Dp(1 1)Gr/+ and a wild type strain in Drosophila melanogaster has been studied by ³H-thymidine autoradiography. Three parameters-labeling frequency, labeling intensity and labeling pattern—have been used to characterize the replication behavior of late labeling spots in the distal part (1A–9A) of the X chromosomes for both genotypes. — Differences in the labeling frequency between homologous subdivisions in both genotypes have been found. Changes of the DNA replication behavior are also indicated by the comparison of labeling patterns in both Drosophila strains. Furthermore, in comparable replication phases the labeling intensities of the Dp(1 1)Gr/+ subdivisions are different from those of the homologous subdivisions in +/+ chromosomes, even where the different DNA amount of both genotypes is taken into consideration.     

Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using a BrdU treatment

Replication times for all important chromosome bands, of both types R and Q (277 structures) are analysed. — The R-bands form a group of structures whose DNA replicates during the early S-phase, while the DNA situated in the Q-bands replicates during the late S-phase. — There may not exist overlapping between replication times of these two types of structures. — The widest R-bands are those which are the earliest to replicate; in general, the most intense Q-bands are those which are the latest to replicate. Especially among these last ones, a certain asynchronism exists between the replication times. Finally the heterochromatin of chromosomes 1, 16 and Y and of the short arms of the acrocentrics could contain two types of DNA which replicate at different times.     

Mimosine Arrests the Cell Cycle after Cells Enter S-Phase

-Mimosine (β-N-[3-hydroxy-4-pyridone]-α-aminopropionic acid)—a rare amino acid derived fromMimosaandLeucaenaplants—arrests cells reversibly late during G1 phase or at the beginning of S-phase. If mimosine were to arrest cells immediately before S-phase, it would provide a superb tool for the investigation of the initiation of DNA synthesis. Therefore, we reexamined the point of action of mimosine. Mitotic HeLa cells were released into 200 μMmimosine and grown for 10 h to block them, before the cells were permeabilized and the amino acid removed by washing them thoroughly. On addition of the appropriate triphosphates, DNA synthesis—measured by the incorporation of [³²P]dTTP—began immediately; as it is known that such permeabilized cells cannot initiate DNA synthesis but can only resume elongating previously initiated chains, mimosine must arrest after DNA synthesis has begun. Moreover, cells grown in mimosine assembled functional replication factories—detected by immunolabeling after incorporation of biotin–dUTP—that were typical of those found early during S-phase. Disappointingly, it seems that mimosine—like aphidocolin—blocks only after cells enter S-phase.     

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

Differential reactivity in mammalian chromosomes

Leucocyte cultures were treated with both ³H-thymidine and low temperature. Leucocyte cells were pulse labeled with ³H-thymidine for 15 to 20 minutes, and then placed in nonisotopic medium for 0, 1, 2, 3 and 4 hours respectively. Each culture was immediately treated with low temperature at 0–3° C for 24 hours. No metaphase chromosome were labeled at 0 and 1 hour after reincubation. Labeled metaphases were first observed after 2 hours of reincubation (3.9%); they increased after 3 hours (57%) and 4 hours of reincubation (39%). Labeled anaphases or telophases were also detectable in increasing proportions after 4 hours. Cell division proceeds very slowly through metaphase at low temperature. After labeling in the final 15 to 20 minutes of the S-period, one X-chromosome usually showed the late-replicating pattern. Label was found in the special segments of the X-chromosomes, X_E–a, X_L–a and X_L–b. Late-replicating regions in autosomes coincide more or less with the special segments. Differential reactivity in human chromosomes by low temperature was suggested to take place during the final part of G₂ after DNA synthesis.     

Die DNS-Synthese in den Chromosomen von Rattus norvegicus während der letzten Stunde der S-Phase

Zusammenfassung An fetalen Zellen der Laborratte wurde der Einbau von ³H-Thymidin in einzelne Chromosomensegmente nach Dauermarkierung quantitativ mittels Kornzählung untersucht. Der ausgewertete Zeitraum umfaßt die letzten 60 min der S-Phase. In diesem Intervall wurde ein distinktes Muster der DNA-Synthese auf den einzelnen Chromosomenabschnitten beobachtet (X-bis Z-Phase). Sehr hohe Syntheseaktivitäten zeigen die Satelliten der Chromosomenpaare Nr. 3 und Nr. 19 sowie die kurzen Arme der Paare Nr. 13 und Nr. 14. Die Aktivitätsmuster der Autosomen stimmen in beiden Geschlechtern im Wesentlichen überein.Das Y-Chromosom ist spät-replizierend und hat auch in der Z-Phase noch einen signifikant erhöhten ³H-Thymidineinbau. Die X-Chromosomen der Ratte sind, entgegen früheren Vermutungen anderer Autoren, nicht die längsten Chromosomen der Gruppe XX, 4–10, sondern das 5. Paar dieser telocentrischen Chromosomen. Mittels Heterochromatinfärbung konnte dieser Befund bestätigt werden.

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DNA Synthesis in the chromosomes of Rattus norvegicus during the Last Hour of the S-Phase

A quantitative analysis of DNA synthesis by means of grain counting after continuous labeling was made on female and male cells of the laboratory rat. Mitoses labeled in the last hour of the S-phase were analysed and the activity sites over individual chromosome segments were plotted. — A non-random distribution of label was detected in this final interval. Very high synthesis activities were observed on the satellites of chromosome No. 3 and No. 19 as well as on the short arms of No. 13 and No. 14. The labeling patterns of the autosomes are corresponding for both sexes. — The Y chromosome is late replicating and shows a relatively heavy label even in the Z interval. In consequence of the labeling pattern in female cells it was concluded that the X chromosomes are not the longest of their group of telocentric chromosomes. In cells exposed to ³H-thymidine during the X and Y interval a prominently labeled putative X chromosome was observed standing between 8th and 10th position in group XX, 4–10. This is in coincidence with results obtained by heterochromatin staining.

     

Initial phases of DNA synthesis in Drosophila melanogaster

Replication patterns of the X chromosomes and autosomes in D. melanogaster male and female larvae during the discontinuously labeled initial and end phases of DNA synthesis were compared. In female larvae X and autosomes behaved correspondingly during all the replication stages. In males, however, the X chromosome shows a differential replication behavior from that of the autosomes already during the discontinuously labeled initial stage.—In those nuclei of both sexes, in which the autosomes correspond in their initial replication patterns, significantly more labeled regions are to be found over the male X than over the female X. The complementary behavior during the end phases (Berendes, 1966), i.e. the reverse of that above, leads to an earlier completion of the replication cycle in most of the labeled regions of the male X chromosome. The differential replication revealed in the autoradiograms is interpreted as a consequence of the polytene structure in giant chromosomes.     

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.     

Autoradiographic studies of the human Y chromosome

An autoradiographic analysis (using continuous labeling with tritiated thymidine) was made on 317 cells from four normal males. The labeling pattern of the Y chromosome was compared to the first and the last chromosomes to complete replication as well as to G21–22. The Y chromosome was never found to be the last chromosome in the cell to complete replication. Instead, it completed DNA synthesis relatively early (usually among the first 10 chromosomes) but had a distinctively heavy label during the earliest stages of late-S. In 51% of those cells with one labeled G+Y chromosome, a G21–22 was labeled and the Y was not.—It was concluded, therefore, that the human Y chromosome is not a late-replicating chromosome but terminates replication earlier than most of the autosomes. In addition, the Y chromosome cannot be distinguished from the G chromosomes on the basis of a consistent and differential labeling pattern.Supported by USPHS Grant GM 15361.     

The Role of MRN in the S-Phase DNA Damage Checkpoint Is Independent of Its Ctp1-dependent Roles in Double-Strand Break Repair and Checkpoint Signaling

The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.     

Variations in the length of S-phase related to the time cells are blocked at the G1-S interface

The inhibition of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine decreases the duration of S-phase of synchronously growing Chinese hamster cultures. — The observed drug effects are discussed in relation to an alteration of programmed DNA replication.     

The behaviour of fragile X and other aberrations during recovery from low folate conditions

Whole blood from two mentally retarded fra-X brothers was grown in low folate medium where fra-X expression was enhanced. Bromodeoxyuridine was added to mitigate the low folate conditions and metaphases were sampled sequentially, and stained for replication banding, through one cell cycle of recovery. — The replication bands allowed detailed analysis of the cell cycle and the allocation of individual cells to precise sub-phases. Various classes of fra-X and all other types of chromosomal aberrations were scored in these classified cells. — The fra-X does not conform in morphology to any of the known simple chromatid intrachange types, which were often present within the same cells, but the subsequent fall in frequency once bromodeoxyuridine was added closely paralleled that of the conventional aberrations. — Normal folate level frequencies of fra-X are restored by the time early S-phase cells (subphase SkI) reach metaphase. When sub-phased cells are rearranged in true chronological sequence, there is a suggestion of a sudden fall in frequency between SkII–III (about 70% of the transit of S). This suggests that the critical point for low folate enhancement occurs in this region of the S-phase. This is somewhat earlier than the band-appearance distribution curve for Xq27 which lies within subphase SkIV.     

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.     

Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed

A number of mammalian genes are expressed from only one of the two homologous chromosomes, selected at random in each cell. These include genes subject to X-inactivation, olfactory receptor (OR) genes, and several classes of immune system genes. The means by which monoallelic expression is established are only beginning to be understood. Using a cytological assay, we show that the two homologous alleles of autosomal random monoallelic loci differ from each other in embryonic stem (ES) cells, before establishment of monoallelic expression. The Polycomb Group gene Eed is required to establish this distinctive behavior. In addition, we found that when Eed mutant ES cells are differentiated, they fail to establish asynchronous replication timing at OR loci. These results suggest a common mechanism for random monoallelic expression on autosomes and the X chromosome, and implicate Eed in establishing differences between homologous OR loci before and after differentiation.