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.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.

The genetically inactive, late-replicating human female X chromosome can be effectively distinguished from its more active, earlier-replicating homologue, when cells are grown according to the appropriate BrdU-33258 Hoechst protocol. Results obtained from a fluorescence analysis of DNA replication in X chromosomes are consistent with those from previous autoradiographic studies, but reflect additional sensitivity and resolution offered by the BrdU-Hoechst methodology. Both qualitative and quantitative differences in 33258 Hoechst fluorescence intensity, reflecting alterations in replication kinetics, can be detected between the two X chromosomes in female cells. The pattern of replication in the single X chromosome in male cells is indistinguishable from that of the early female X. Intercellular fluctuations in the distribution of regions replicating early or late in S phase, particularly with reference to the late female X, can be localized to structural bands, suggesting multifocal control of DNA synthesis in X chromosomes.     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

Cytogenetic and biochemical investigations on fibroblast cultures and clones with one and two active X chromosomes of a 69,XXY triploidy

Bromodeoxyuridine replication patterns showed that fibroblasts from a 69,XXY triploidy carried either one or two early replicating X chromosomes. The activity of alpha-galactosidase A measured in single cells fell into two classes with a ratio of 1:2. Dilute plating produced clones of both types with the activity of alpha-galactosidase A corresponding to the number of active X chromosomes. To our knowledge, this is the first report on clones of a triploidy with different numbers of active X chromosomes, and on a gene-dosage effect of an X-linked gene using triploid cells with one active X as control.     

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.     

Late replicating X chromosomes in human triploidy.

The status of X-chromosome replication was studied in twenty-seven 69,XXY and nine 69,XXX human triploids in which the parental origin of the additional haploid set was known from the study of chromosome heteromorphisms. Among the 69,XXY triploids, fourteen had no late replicating X, two had one late replicating X in all cells examined, and eleven had two populations of cells, one with late replicating X chromosome, and one without any. Among the 69,XXX triploids, four had a single late replicating X, and five had two populations of cells, one with one late replicating X, and one with two late replicating X chromosomes. There was no correlation between the parental origin of the triploidy and the type of X-chromosome inactivation. However the number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue when compared with those grown from extra-embryonic tissue. In cultures derived from extra-embryonic tissue there was a significant correlation between the gestational age of the sample and the proportion of late replicating X chromosomes. The older the specimen, the greater the number of late replicating X chromosomes.     

Allocyclic early replicating X chromosome in mice: Genetic inactivity and shift into a late replicator in early embryogenesis

The allocyclic X chromosome in early female mouse embryos undergoes DNA replication either late or early in the S phase. Earlier studies indicated that the early-replicating X chromosome is restricted to the trophectoderm and primitive endoderm cell lineages in which the allocyclic X is almost exclusively paternal in origin. There has been, however, no compelling evidence for the genetic inactivity of the early-replicating X chromosome and a shift from early to late replication or vice versa. The present study employing a combination of 3H-thymidine autoradiography and BrdU labeling-acridine orange fluorescence staining in day-6 female mouse embryos found that the early-replicating X chromosome can change directly into a late-replicating one. The activity state of the early-replicating X chromosome was examined by electrophoretic determination of the X linked enzyme, phosphoglycerate kinase (PGK-1), in tissues isolated from 6.0-day and day-8.5 Pgk-1a/Pgk-1b embryos. Only the maternally derived Pgk-1 allele was expressed in the proximal endoderm and extraembryonic ectoderm of 6.0-day and the chorion of 8.5-day embryos. Thus, the early-replicating, paternally derived X chromosome found in about 70%-80% of the cells in these tissues seems to be repressed like the late-replicating one.     

DNA methylation of the X chromosomes of the human female: an in situ semi-quantitative analysis

We present an in situ semi-quantitative analysis of the global DNA methylation of the X chromosomes of the human female using antibodies raised against 5-methylcytosine. The antibodies were revealed by immunofluorescence. Images were recorded by a CCD camera and the difference in intensity of fluorescence between active (early replicating) and inactive (late-replicating) X chromosomes was measured. Global hypomethylation of the late-replicating X chromosomal DNA was observed in three cases of fibroblast primary cultures that were characterized by numerical and structural aberrations of the X chromosomes [46,X,ter rea(X;X), 48,XXXX and 46,X,t(X;15)]. In these cases, the difference between early and late-replicating X chromosomes was significantly greater than the intrametaphasic variations, measured for a pair of autosomes, that result from experimental procedures. In cells with normal karyotypes, the differneces between the two X chromosomes were in the range of experimental variation. These results demonstrated that late replication and facultative heterochromatinization of the inactive X are two processes that are not related to global hypermethylation of the DNA     

Evidence for a relationship between DNA methylation and DNA replication from studies of the 5-azacytidine-reactivated allocyclic X chromosome

We examined the sequence of DNA synthesis of the human active, inactive and reactivated X chromosomes in mouse-human hybrid cells. The two independent reactivants, induced by 5-azacytidine (5-azaC), expressed human hypoxanthinephosphoribosyl transferase (HPRT), and one also expressed human glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK). Restriction enzyme analysis of DNA methylation at the re-expressed loci revealed hypomethylation of CpG clusters, that characterizes the relevant genes on the active X. The transfer of active and inactive X chromosomes from the native environment of the human fibroblast to the foreign environment of the hybrid cell did not affect the specific replication sequence of either human X chromosome. The silent X chromosome when reactivated, remained allocyclic, and the first bands to replicate were the same as prior to reactivation. In one reactivant, however, further progression of replication was significantly altered with respect to the order in which bands were synthesized. This alteration in the replication of the silent X following 5-azaC-induced reactivation suggests that DNA methylation may modulate the replication kinetics of chromosomal DNA.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Derepression with decreased expression of the G6PD locus on the inactive X chromosome in normal human cells

Studies of a unique clone of skin fibroblasts from a normal 46 XX female reveal that the G6PD locus on the inactive X chromosome has been derepressed. The reactivation event occurs spontaneously, and is associated with normal karyotype, including the presence of a late-replicating X chromosome. Analysis of mouse-human hybrids with the relevant chromosome provides evidence that the derepressed locus is on the inactive X, and that reactivation is not extensive (the PGK locus is not derepressed). Nor is any general change in DNA methylation of this chromosome detectable with Hpa II and an X-specific DNA probe. Studies of the glucose-6-phosphate dehydrogenase phenotype in these heterozygous cells indicate that the reactivated X produces only half the enzyme subunits as are produced by the active X. Although this dosage difference may be related to the mutational event responsible for derepression of the locus, these observations along with other evidence suggest that loci on the inactive X, when expressed, have less activity than corresponding loci on the active X.     

Cytogenetic evidence for the absence of an inactivated X chromosome in a human female (XX) breast carcinoma cell line

Summary Barr body staining procedures were applied directly to the chamber slide cultures of female amniotic cells, WI38 fibroblasts, normal female kidney cells, and a human breast carcinoma cell line, Elco. A high frequency of Barr bodies was found in all the normal female control cells; however, no Barr bodies were observed in the Elco cells. By trypsin G-banding analysis, two normal X chromosomes were identified in all Elco cells. The late DNA replication pattern of the cell line was then studied with the terminal BrdU pulse method. Both X chromosomes in the Elco cell line were found to be euchromatic with a characteristic R-banding pattern; no late-replicating X chromosome was observed. The absence of both a Barr body and a late-replicating heterocyclic X chromosome provides strong cytogenetic evidence that an inactivated X chromosome is absent in the human breast carcinoma cells bearing two X chromosomes.     

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2'-deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment-strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Sister chromatid differentiation and isolabeling of chromosomes

Summary Isolabeling observed during sister chromatid differentiation (SCD) was studied from human skin fibroblasts by the fluorescence-plus-Giemsa (FPG) technique. Bromodeoxyuridine (BrdU) was fed to exponentially dividing cells for 52 h to enable completion of two consecutive cycles of DNA replication. During this period, the late-replicating regions of some chromosomes were able to go through three replication cycles. These chromosome regions had evidently incorporated BrdU bifiliarly in both chromatids and hence, on staining with FPG, appeared isostained (isolabeled). Thus, incubation of exponentially dividing cells with BrdU for a period longer than that required for two cell cycles appears to be a suitable method for revealing the late-replicating regions of the genome, such as the X chromosome in a human female, as isolated.In another experiment with Indian muntjac chromosomes, isolabeled segments were darkly stained, which suggested unifilar incorporation of BrdU. In this case, unequal crossing-over or an unequal distribution of thymine residues probably is responsible for the isolabel.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.