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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In normal diploid fibroblasts of the mouse, 3T3-, SV-3T3-, and Meth A-cells, the chromosome replication patterns were studied by a bromodeoxyuridine (BrdU)-labelling technique. SV-3T3 is a subline of 3T3 transformed by SV 40 and Meth A is a permanent cell line from Balb c transformed by methylcholanthrene. The use of 1 h thymidine pulses permits high resolution of the S-phase after partial synchronization of the cells at G1/S in an otherwise BrdU-substituted S-phase. It could be shown that the autosomal heterochromatin of the mouse (Mus musculus) starts replication during the early S-phase (R-band replication), continues while R-band chromatin finishes, and still replicates when G-band chromatin starts. The heterochromatin finishes before the majority of G-bands have been replicated. There is no fundamental difference in the course of chromosome replication between the different cell lines studied here. It is concluded that there are no obligate changes in the course of the S-phase linked to the process of transformation.
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  • 2
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: 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.
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  • 3
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Protocols are compared demonstrating sister chromatid differentiation (SCD) in human amniotic fluid (AF) cells with and without partial synchronization. Partial synchronization both with an excess of 5-bromodeoxyuridine (BrdU) and an excess of thymidine leads to an increase of metaphases with SCD. Compared with unsynchronized cells, the rate of sister chromatid exchanges (SCE) is not increased. Studies on the late replicating X chromosome of female cells showed that the addition of mitomycin C (MMC) after releasing the thymidine block preferentially induces SCEs in late replicating regions. The partial synchronization with thymidine surplus provides a good basis for SCE experiments with AF cells and facilitates the prenatal diagnosis of diseases characterized by changes in the SCE rate.
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  • 4
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A psychomotor-retarded infant with minor dysmorphic signs and a karyotype 46.XY,r(21)mat in lymphocytes is reported. The mother is phenotypically normal but shows the same unstable r(21). This is another case demonstrating that a chromosomal aberration does not necessarily lead to infertility by meiotic failure. Nevertheless, segregation of ring chromosomes is problematic for two reasons: mitotic problems of the ring structure itself and synaptic difficulties during the pachytene stage.
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  • 5
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A series of fibroblasts from patients with numerical or structural aberrations of the X chromosome were scored for the amount of mRNA of ribosomal protein S4 (RPS4X). Haplo-insufficiency of this gene has been reported previously to be a possible cause of Turner syndrome. Our results show that the transcription rate of RPS4X correlates with the number of gene copies. This confirms earlier findings indicating that this gene escapes X inactivation. In addition, we demonstrate that this applies to structurally aberrant X chromosomes. Our results show that RPS4X does not give rise to a type of haplo-insufficiency in these cases, because it escapes inactivation, even on structurally aberrant X chromosomes from patients with Turner syndrome. We therefore assume that RPS4X is not the most prominent candidate gene for Turner syndrome.
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  • 6
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The “pseudoautosomal expression” of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.
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  • 7
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chromosome replication in the last premeiotic S-phase of male mammals has been previously studied by [3H]thymidine autoradiography and by a 5-bromodeoxyuridine (BrdU)/Giemsa technique. We used a recently developed BrdU-antibody technique (BAT) in this study. The following conclusions were drawn: (1) The replication patterns observed are similar to that of somatic cells. (2) The heterochromatin starts replication in early S-phase. (3) The euchromatic part of the X chromosome of the male Chinese hamster replicates together with the autosomes and therefore behaves isocyclicly and not allocyclicly as hitherto assumed. Hence, genetic inactivity of the X chromosome may be brought about by a mechanism different from that in somatic cells.
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  • 8
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chinese hamster ovary (CHO) and HeLa cells were successively pulse labelled at 1-h intervals after the cultures were synchronized at the end of G1 (monitored by flow cytometry). The metaphases analysed afterwards showed R-type replication patterns after 1-h pulses during the early S-phase (SE; from h 1–5 after release) and replication of G− and C−bands in late S-phase (SL: from h 6–8 after release). The transition from SE to SL is abrupt, constituting a sudden switch of replication between different types of chromatin as has been described for human lymphocytes. The differences between these two cell lines and earlier results reported on a V79 Chinese hamster cell line and on normal diploid human and Chinese hamster fibroblasts are discussed.
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  • 9
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.
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  • 10
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.
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