BrdU-33258 Hoechst analysis of DNA replication in human lymphocytes with supernumerary or structurally abnormal X chromosomes

BrdU-33258 Hoechst techniques have been used to characterize DNA replication patterns in lymphocytes from human females with supernumerary or structurally abnormal X chromosomes. Fluorescence analysis permits identification of late replicating X chromosomes in a very high proportion of cells and affords a high resolution method for determining the interchange points of X-X and X-autosome translocations. Asynchrony among terminal replication patterns of multiple late replicating X chromosomes within an individual cell can occasionally be demonstrated. The arms of isochromosomes usually exhibit symmetrical fluorescence patterns, with replication terminating in bands Xq21 and Xq23 (predominant pattern) or in bands Xq25 and Xq27 (alternative pattern) in both arms. In the vast majority of lymphocytes containing a balanced X-13 or X-19 translocation, the normal X is late replicating. However, DNA synthesis in the translocation products occasionally appears somewhat delayed relative to that expected for an early replicating X, consistent with possible position effects on replication kinetics.     

Different patterns of X chromosome inactivity in lymphocytes and fibroblasts of a human balanced X; autosome translocation

Summary In lymphocytes of a human female carrier of a balanced X;3 translocation, 46,X,t(X;3)(q28;q21), late replication of the structurally normal X chromosome only was previously described (de la Chapelle and Schröder 1973). We have now confirmed this finding using a fresh blood sample. Examining the chromosomes of this individual in fibroblasts we observed that either the normal X or the Xq+ chromosome could replicate late and show inactivity after fusion with heteroploid mouse cells. The replication patterns of chromosomes in human X;autosome translocations have so far almost exclusively been analyzed in lymphocytes. Our findings stress that results based on these cells are not representative for all cell types.     

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.     

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.     

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.     

Tandem duplication dup(X)(q13q22) in a male proband inherited from the mother showing mosaicism of X-inactivation

Summary An aberrant X chromosome containing extra material in the long arm was observed in a psychomotoric retarded boy and his healthy, short-statured mother. The proband showed generalized muscular hypotony, growth retardation, and somatic anomalies including hypoplastic genitalia and cryptorchism.Chromosomal banding techniques suggested a tandem duplication of the segment Xq13Xq22.In the mother the vast majority of lymphocytes showed late replication of the aberrant X chromosome. Some of her cells, however, contained an apparently active aberrant X. Both the early- and late-replicating aberrant X exhibited late replication patterns very similar to those described for normal X chromosomes in lymphocytes. Asynchrony of DNA replication among the two segments Xq13Xq22 in the dup(X) was never observed.We consider that the clinical picture of the proband is caused by an excess of active X material.     

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.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Replication kinetics of X chromosomes in fibroblasts and lymphocytes

Summary The kinetics of replication for early and late replicating X chromosomes in karyotypically normal fibroblasts and lymphocytes was studied using terminal bromodeoxyuridine (BrdU) treatment followed by Hoechst/light/Giemsa staining. Although the order of band appearance differs between the two tissues, the programme (order and interval between band appearances) for early replicating bands (dark R-bands) is identical in the two homologues. This is probably also the case for later replicating bands (dark G-bands) though the criteria for derermining mean band appearance times are less reliable for these bands when terminal BrdU treatment is used. This means that the late X has a delayed start but thereafter proceeds at the same pace as its early counterpart.     

Kinetic heterogeneity of the replication of genetically inactive X chromosome in human cells

Kinetics of DNA replication in genetically non-active X chromosome was studied in peripheral lymphocytes and skin fibroblasts from four phenotypically normal women and one fetus using BrdU 33258 Hoechst-Giemsa techniques. The localization of the earliest replicated chromosomal segment was shown to be unstable, varying from cell to cell in both lymphocytes and fibroblasts of all persons examined. Several variants of replication sequence in the X chromosome were found in both types of cells. The variants revealed were classified, according to Willard. The statistically significant differences in replication sequence were found between blood lymphocytes and skin fibroblasts in two individuals. The problem of tissue specificity in replication kinetics of the genetically non-active X chromosome is discussed.     

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes

Summary X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.     

Inherited X-chromosome inverted tandem duplication in a male traced to a grandparental mitotic error.

A male infant was referred for cytogenetic evaluation because of dysmorphic features and developmental delay. In both lymphocytes and skin fibroblasts, a modal number of 46 chromosomes was obtained with an obvious elongation of the long arm of the X chromosome (Xq+). Studies of seven members in 3 generations of this family showed that the proband's mother, sister, and maternal grandmother were phenotypically normal carriers of this abnormal X chromosome. High resolution GTG- and RBG-banding defined the extra chromatin material as an inverted duplication of Xq21----Xq24. This was supported by an approximate twofold increase in alpha-galactosidase A activity, localized to Xq21----q24, observed in the proband's lymphocytes and fibroblasts. BrdU-incorporation studies of the mother's lymphocytes showed the abnormal X to be late replicating in all 100 cells studied and normal alpha-galactosidase A levels. Cytogenetic analysis of the maternal grandmother revealed cytogenetic mosaicism with one cell line containing the abnormal X (37%), and the other, a normal female karyotype (63%). This family is instructive since: (1) it represents only the second case of a dysmorphic male demonstrating a confirmed interstitial partial Xq duplication, and (2) the origin of this familial structural rearrangement has been traced to a grandparental mitotic error.     

Chronology of the replication of sex chromosome bands in lymphocytes of normal subjects and patients]

The replication sequence of the bands carried by chromosomes X and Y has been studied in normal individuals and in patients with structural abnormalities of the X. By comparing the segment with that of the autosomal bands (which had been previously studied), it was shown that the normal early X replicates in early X-phase for its R-bands and in late S-phase for its Q bands. The late X replicates entirely in late S-phase, and the sequence of band replication is not as stringent as for the early X and the autosomes. The study of fourteen cases of anomalies of chromosome X in females showed the following: in balanced reciprocal X-autosome translocations the rearranged X most often replicates early and the normal X late. Both show a normal replication sequence of their bands. In non-balanced X-autosome translocations, inactivation of the autosome fragment attached to the AUTOSOME FRAGMENT ATTACHED TO THE X may take place. In Xq- or in ter rea (X;X) (pter;pter), band p22 has a delayed replication. In iso-Xor Xp-, the long-arm-band sequence of replication shows a variation comparable to that of the late X in fibroblasts. These replication modifications are likely to induce partial inactivations or changes in activity which correspond to the so-called position effect in Drosophila.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

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.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Pattern of late DNA replication of the allocyclic X chromosome in Cebus apella and Leontopithecus rosalia chrysomelas fibroblasts

The pattern of late DNA replication in the allocyclic X chromosome has been studied in the primary fibroblasts of two neotropical primates (Cebus apella and Leontopithecus rosalia chrysomelas). A comparison with previous reports showed a pattern identical with that of (1) the allocyclic X chromosome of human fibroblasts, and (2) the allocyclic X chromosome of rhesus and Cebus lymphocytes. Our results show that at least in one species (C. apella), and probably in rhesus and Leontopithecus, there is no tissue-specific difference between the late DNA replication patterns of the allocyclic X chromosome as there is between human lymphocytes and fibroblasts.     

DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique.

A technique is described for visualizing late-replicating regions by a Hoechst 33258-Geimsa-staining procedure combining the techniques of Latt (1973) and of Perry and Wolff (1974). The advantages are two-fold: distinct bands are obtained and many possible mistakes and interpretation difficulties with autoradiography are avoided. The time sequence of late-replication patterns (excepting C-group chromosomes) has been established in human fibroblasts of adults, and these results have been compared with three of four different cell types from amniotic fluid. No significant differences in late-replication patterns and time sequence of the different cells could be discovered. As expected, the replication patterns are in good concordance with the patterns of G- and Q-bands. Some exceptions are described.     

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.