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.     

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.     

Early replication banding in <Emphasis Type="Italic">Leporinus</Emphasis> species (Osteichthyes,Characiformes) bearing differentiated sex chromosomes (ZW)

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2′-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.     

Cytologic evidence for three human X-chromosomal segments escaping inactivation

Summary Early replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).     

What causes the abnormal phenotype in a 49,XXXXY male?

Summary The chromosome replication pattern of a man with 49,XXXXY was analyzed using ³H-thymidine and autoradiography as well as BrdU and acridine orange. The former technique showed a highly irregular replication pattern; the latter revealed one early replicating X chromosome, and the other three more or less asynchronously replicating. Two hypotheses seem to explain best the abnormal phenotype of males with an XXXXY sex chromosome constitution: (1) The number of the always active regions (tip of Xp) and of the possibly always active regions (the Q-dark regions on both sides of the centromere) is increased from one to four. (2) The replication pattern of the late-replicating X chromosomes is highly asynchronous, which might affect the phenotype. The possibility that more than one X chromosome might remain active in some cells, an even more abnormal and obviously deleterious situation, is still open.     

大熊猫染色体晚复制带研究

以培养的大熊猫外周血淋巴细胞为实验材料,在细胞培养终止前４ｈ加入ＢｒｄＵ（终浓度为１０μｇ／ｍｌ培养基）,对复制的染色体ＤＮＡ进行ＢｒｄＵ标记。掺入ＢｒｄＵ的染色体经吖啶橙（０．０５％）处理、紫外光照射、Ｇｉｅｍｓａ染色后,可在染色体上获得清晰的复制带纹。根据众多分裂相所显示的不同复制带型,可初步确定大熊猫每一染色体独特的晚复制带纹。在雌性个体的两个Ｘ染色体中,一条Ｘ染色体复制明显落后于另一Ｘ染色体,尤其在迟复制Ｘ染色体长臂近着丝粒区显现出较宽的晚复制带纹。     

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.     

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.     

Heterozygous female carriers of the marker-X-chromosome: IQ estimation and replication status of fra(X)(q)

Summary The IQ levels of 18 female carriers with the marker X chromosome were evaluated, and cytogenetic studies after BrdU incorporation were performed. A highly significant correlation between mental capacity and replication pattern of the X chromosomes could be demonstrated. Heterozygous females with normal intelligence showed a clear tendency to carry the fragile site at the late replicating X chromosome, while other female carriers with lower intelligence or mental impairment expressed their fragile site mainly with the early replicating X chromosome. This observation could be interpreted as an expression of Lyonisation.     

Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

家蚕染色体复制区带的初步显示

该文采用家蚕Bomoyx mori活体注射BrdU结合FPG(fluorochrome photolyusis Giem-sa)显带方法,以生殖腺为材料,成功显示出家蚕有丝分裂中期染色体复制带。由于处于S-期的细胞有早有晚,且同一细胞DNA各片段的复制亦有先后,因此BrdU掺入DNA合成的时间也有所不同,从而可产生出早、中、晚复制带型。BrdU掺入时间早,则会在家蚕部分染色体上出现大面积浅染带纹的早复制带。每一染色体皆有其独特的带纹特征,据此可初步将它与其它染色体相互区分;随着BrdU掺入时间的推后,染色体上会出现深浅交替、丰富的带纹,即中复制带型;至S-期DNA合成晚期掺入BrdU,最终染色体出现以深染带纹为主,浅染带纹仅出现于少数染色体的中部、近中部或端部的晚复制带。     

Prometaphase banding of human chromosomes with basic fuchsin

Summary A technique is described for the production of detailed and richly contrasting G-band patterns in human prometaphase chromosomes with the aid of the triphenylmethane dye basic fuchsin. The usefulness of this method is illustrated by its application for the precise analysis of two chromosome 11 rearrangements. It is also demonstrated that high-resolution banding with basic fuchsin can reveal bands not present in the international standard idiogram of human prophase chromosomes (ISCN 1981). The technique described can also be used for easy recognition of the late replicating X chromosome, which stains darker than its early replicating homologue. A preliminary analysis of the late replicating X chromosomes in a 49,XXXXY individual suggests that the three supernumerary X chromosomes do not necessarily replicate synchronously.     

Sex chromatin and X chromosome replication patterns and incidence of sex chromatin in canine cell cultures

In order to provide evidence as to whether sex chromatin (SC) of interphase cells is equivalent to the late replicating X chromosome in female mammalian cells, time-lapse cinephotometric and autoradiographic methods were used to give precise data for comparison of the DNA replication patterns of SC with that of each of the X chromosomes throughout the S period. Canine kidney epithelial cells were selected because they have distinct large metacentric X chromosomes and typical SC. Time-lapse cinephotometry was used to avoid possible alteration of DNA synthesis by chemical cell synchronization agents. Determination of the incidence of SC during the stages of the cell life cycle of proliferating cells of the same origin was performed in order hopefully to clarify conflicting reports on the subject. Our results clearly show that time and intensity of the SC replication throughout S period is like that of the late replicating X chromosome and unlike that of the early replicating X chromosome. The incidence of SC in proliferating cells in culture was found to vary with the stage of the cell life cycle, increasing with increasing postmitotic interval — least in G1, greater in S, and greatest in G2. The SC incidence increased strikingly from G1 to S and a less marked increase was observed between S and G2.     

Chromosome replication patterns in the Djungarian hamster (Phodopus sungorus)

Patterns of early and late replication in the individual chromosomes of the Djungarian hamster (Phodopus sungorus) have been studied using the techniques of Giemsa staining suppression when bromodeoxyuridine is incorporated into the DNA. — Late replicating autosome regions correspond to G-band regions, early replication regions are less clearly demarcated but correspond to R-band regions plus some G-band zones. In part this reduction in sharpness of early replication bands may be due to the fact that nearly all metaphase G-bands contain R-band material since they are compounded from blocks of sub-G bands. — The long arm of the X chromosomes in the female differ in the start time of synthesis but are rarely separable at the close of S. There are no differences between the short arms. In the male, Y starts very late but finishes about the same time as the X which behaves like the early replicating X of the female.Visiting worker from Department of Biological Sciences, Sambulpur University, Burla 768017, India     

Patterns of replication in the neo-sex chromosomes ofDrosophila nasuta albomicans

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species ofDrosophila.     

Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine

Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: initially the BrdU is incorporated during the first part of the S-phase and then it blocks the cells at mid-S-phase. Within the cell cycle, mid-S-phase appears to be the most vulnerable time to various blocking agents. To differentiate the regions of BrdU incorporation from those that have not been substituted, the fluorescence-photolysis-Giemsa (FPG) technique was applied as modified for horse chromosomes. This dynamic technique, which produces many prometaphase and prophase chromosomes showing very sharp G-bands, is certain to enhance the accuracy of cytogenetic analysis and aid in the standardization of equine chromosomes.     

Studies on early replication patterns in the marsupial Sminthopsis crassicaudata: no evidence for XY replication homologies

We present here the first detailed replication banding study of a marsupial species using the BrdU-replication technique. A comparison of the structural and replication bands of the chromosomes of Sminthopsis crassicaudata clearly demonstrates that the replication behavior is the same as the described for the chromosomes of eutherians. The early replicating segments correspond to R-bands, whereas the late-replicating regions tend to be situated within Q- and C-bands. Use of this technique clearly reveals an early and late replicating X chromosome. The very small Y chromosome can be subdivided into two replication segments, but no replication homologies can be demonstrated between the X and Y chromosomes of S. crassicaudata.     

Ultrastructure,meiotic behavior,and evolution of sex chromosomes of the genus Ellobius

The whole-mount SC preparations from males of three species of the genus Ellobius (Ellobius fuscocapillus, Ellobius lutescens), and Ellobius tancrei were studied by electron microscopy. In the males of Ellobius fuscocapillus, behavioral peculiarities of the sex bivalent (viz. the normal male heterozygosity) are characterized by early complete desynapsis of sex chromosomes (X, Y), occurring at late pachytene-early diplotene. The karyotype of species Ellobius lutescens is unique for mammals. In both sexes it is characterized by an odd number of chromosomes (2n=17). At prophase I the unpaired chromosome 9 is not involved in synapsis with other chromosomes and forms a sex body at the end of pachytene.The complete Robertsonian fan has been described for superspecies Ellobius tancrei. As shown on the basis of G-band patterns the male and female sex chromosomes are cytologically indistinguishable.Analysis of whole-mount SC preparations revealed the formation of a closed sex SC bivalent and showed some morphological differences in the axes of sex chromosomes at meiotic prophase I. A number of assumptions are made about the relationship between the behavior of sex chromosomes, their evolution and the sex determination system in the studied species of genus Ellobius.

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Functional and Structural Chromosome Analyses in Autotetraploid Silene latifolia

Recent immunocytological and molecular data show that heterochromaticnuclear regions, both constitutive and facultative, are modifieddifferently (cytosine hypermethylation and histone hypoacetylation)and late replicating, when compared to euchromatin. Intrusiveand/or additive (supernumerary) DNA sequences are often functionallysilenced; this is accompanied by their heterochromatinization.In this work we present a number of karyological studies onautotetraploid female cells of Silene latifolia (syn. Melandriumalbum). Immunofluorescence analyses do not indicate any globaldifferences in DNA methylation, histone H4 acetylation, andchromosome replication patterns which could arise as a consequenceof the duplication of the whole chromosome set of the originaldiploid genome. Similarly, the number of silver-positive nucleoliroughly correlates to the ploidy level. Early replication andH4 hyperacetylation have been detected at all subterminal chromosomeregions. This, together with cDNA in situ hybridization patterns,indicates the localization of gene-rich regions. DNA methylationand chromosome replication patterns, but not histone H4 acetylation,show differences among the four X chromosomes present: one tothree X chromosomes were observed as hypermethylated and/orlate replicating. Taken together, the data demonstrate thatthere is no overall silencing of the additional two sets ofautosomes in the tetraploid cells, but the X chromosomes couldbe subject to an irregular dosage compensation. Copyright 1999Annals of Botany Company DNA methylation, histone acetylation, polyploidy, replication patterns, sex chromosomes, Silene latifolia (syn.Melandrium album ).     

Prenatal diagnosis of a 46,XX,inv(12)pat/47,XX,i(Xq),inv(12)pat

Summary A 46,XX,inv(12)pat/47,XX,i(Xq),inv(12)pat was diagnosed prenatally in a 36-year-old woman whose husband was a known carrier of a pericentric inversion of chromosome 12. The diagnosis was confirmed in fetal tissue. Terminal bromodeoxyuridine (BrdU) labelling demonstrated that in the line with 46 chromosomes one X was late replicating, while one X and the i(Xq) were late replicating in 100% of the cells with 47 chromosomes. We present the first case of this type of sex chromosome mosaicism. Genetic counseling presented difficulties since it was not possible to predict the fetal phenotype.