Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Genes and genomes: Chromosome bands – flavours to savour

The mammalian chromosome is longitudinally heterogeneous in structure and function and this is the basis for the specific banding patterns produced by various chromosome staining techniques. The two most frequently used techniques are G, or Giemsa banding and R, or reverse banding. Each type of stained band is characterised by variations in gene density, time of replication, base composition, density of repeat sequences, and chromatin packaging. It is increasingly apparent that R and G bands, which are complementary to each other, represent separate compartments of the euchromatic human genome, with R bands containing the vast majority of genes. R bands are also more GC-rich, contain a higher density of Alu repeats, and replicate earlier in S phase, than G bands. These properties may be interdependent and may have coevolved.     

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.     

Evolution of differential chromosome banding]

Specific chromosome banding patterns in different eukaryotic taxons are reviewed. In all eukaryotes, chromosomes are composed of alternating bands, each differing from the adjacent material by the molecular composition and structural characteristics. In minute chromosomes of fungi and Protozoa, these bands are represented by kinetochores (Kt- (Cd-)bands), nucleolus organizers (N-bands), and telomeres as well as the euchromatin. In genomes of most fungi and protists, long clusters of tandem repeats and, consequently, C-bands were not revealed but they are likely to be found out in species with chromosomes visible under a light microscope, which are several tens of million bp in size. Chromosomes of Metazoa are usually larger. Even in Cnidaria, they contain C-bands, which are replicated late in the S phase. In Deuterostomia, chromosome euchromatin regions differ by replication time: bands replicating at the first half of the S phase alternate with bands replicating at the second half of the S phase. Longitudinal differentiation in the replication pattern of euchromatic regions is observed in all classes of Vertebrata beginning with the bony fish although the time when it developed in Deuterostomia is unknown. Apparently, the evolution of early and late replicating subdomains in Vertebrata euchromatin promoted fast accumulation of differences in the molecular composition of nucleoproteid complexes characteristic of early and late replicating bands. As a result, the more contrasting G/R and Q-banding patterns of chromosomes developed especially in Eutheria. The evolution of Protostomia and Plantae followed another path. An increase in chromosome size was not accompanied by the appearance of wide RBE and RBL euchromatin bands. The G/R-like banding within the interstitial chromosome regions observed in some representatives of Invertebrates and higher plants arose independently in different phylogenetic lineages. This banding pattern seems to be closer to that of C-banding than to the typical G/R-banding of the mammalian chromosomes.     

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     

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R-and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.     

Replication timing of the human X-inactivation center (XIC) region: correlation with chromosome bands

The human genome is composed of long-range G+C% mosaic structures, which are thought to be related to chromosome bands. Replication timing during S phase is associated with chromosomal band zones; thus, band boundaries are thought to correspond to regions where replication timing switches. The proximal limit of the human X-inactivation center (XIC) has been localized cytologically to the junction zone between Xq13.1 and Xq13.2. Using PCR-based quantification of the newly replicated DNA from cell-cycle fractionated THP-1 cells, the replication timing in and around the XIC was determined at the genome sequence level. We found two regions where replication timing changes from the early to late period during S phase. One is located near a large inverted duplication proximal to the XIC, and the other is near the XIST locus. We propose that the 1Mb late-replicated zone (from the large inverted duplication to XIST) corresponds to a G-band Xq13.2. Several common characteristics were observed in the XIST region and the MHC class II-III junction which was previously defined as a band boundary. These characteristics included differential high-density clustering of Alu and LINE repeats, and the presence of polypurine/polypyrimidine tracts, MER41A, MER57 and MER58B.     

Cytogenetic replication studies on murine T-cell leukemias with special consideration to chromosome 15

Chromosomal replication was studied by means of the BrdU-Höchst-Giemsa-technique in three murine T-cell leukemias with special regard to chromosome 15. In all the three leukemic cell lines the normally early replicating band 15 E was undetectable whereas late replication in this region is completely unaltered. Furthermore, R-banding, the structural homologue of early replication banding, remains unchanged. This observation is interpreted as a shift in the time point of replication during the S-phase of region 15 E, though the exact timing of replication of this region in tumor chromosomes remains unresolved.     

Dual control of replication timing. Stochastic onset but programmed completion of mammalian chromosome duplication.

In mammalian cells, DNA replication proceeds according to a precise temporal order during the S phase, but how this program is controlled remains poorly understood. We analyzed the replication-dependent bromodeoxyuridine banding of chromosomes in Chinese hamster cells treated with the spindle poison nocodazole. In these cells, nocodazole induces a transient mitotic arrest, followed by DNA re-replication without intervening cell division. Nuclear fragmentation is often observed in tetraploid derivatives, and previous studies suggest that replication timing of chromosomes could be affected when they are segregated into different micronuclei. Here we show that the onset of replication is frequently asynchronous on individual chromosomes during the re-replication process. Moreover, fluorescence in situ hybridization analysis revealed that replication synchrony is equally altered in fragmented and non-fragmented nuclei, indicating that asynchronous onset of replication is not dependent on physical separation of the chromosomes into isolated compartments. We also show that the ordered program of replication is always preserved along individual chromosomes. Our results demonstrate that the onset of replication of individual chromosomes in the same nuclear compartment can be uncoupled from the time of S-phase entry and from the programmed replication of chromosome sub-domains, revealing that multi-level controls contribute to establish replication timing in mammalian cells.     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

The hierarchically organized splitting of chromosome bands into sub-bands analyzed by multicolor banding (MCB)

To clarify the nature of chromosome sub-bands in more detail, the multicolor banding (MCB) probe-set for chromosome 5 was hybridized to normal metaphase spreads of GTG band levels at approximately 850, approximately 550, approximately 400 and approximately 300. It could be observed that as the chromosomes became shorter, more of the initial 39 MCB pseudo-colors disappeared, ending with 18 MCB pseudo-colored bands at the approximately 300-band level. The hierarchically organized splitting of bands into sub-bands was analyzed by comparing the disappearance or appearance of pseudo-color bands of the four different band levels. The regions to split first are telomere-near, centromere-near and in 5q23-->q31, followed by 5p15, 5p14, and all GTG dark bands in 5q apart from 5q12 and 5q32 and finalized by sub-band building in 5p15.2, 5q21.2-->q21.3, 5q23.1 and 5q34. The direction of band splitting towards the centromere or the telomere could be assigned to each band separately. Pseudo-colors assigned to GTG-light bands were resistant to band splitting. These observations are in concordance with the recently proposed concept of chromosome region-specific protein swelling.     

A compositional map of the cen-q21 region of human chromosome 21

A compositional map of the centromere and of the subcentromeric region of the long arm of human chromosome 21 was established by determining the GC levels (GC is the molar fraction of guanine+cytosine in DNA) of 11 YACs (yeast artificial chromosomes) covering this 13–14 Mb region which extends from the α-satellite sequences of the C(entromeric) band qll.1, through R(everse) band q11.2, to the proximal part of G(iemsa) band q21. The entire region is made up of GC-poor, or L, isochores with only one GC-rich H1 isochore, at least 2 Mb in size, located in band q21. The almost identical GC levels of the centromeric α-satellite repeats (38.5%), of R band q11.2 (39%), and of G bands (38–40%) provide a direct demonstration that base composition cannot be the only cause of the cytogenetic differences between C, G, and the majority of R bands, namely the H3^- R bands (which do not contain the GC-richest H3 isochores). The results obtained also show that isochores may be as long as 6 Mb, at least in the GC-poor regions of the genome, and support previous observations suggesting that YACs from isochore borders are unstable and/or difficult to clone. Genes and CpG islands are very rare in the GC-poor region investigated, as expected from the fact that their concentration is proportional to the GC levels of the isochores in which they are contained.     

小麦属核型分析和BG染色体组及4A染色体的起源

应用植物有丝分裂染色体标本制备新方法和N—带技术对小麦属(Triticum)9个六倍体种(AABBDD),8个四倍体种(AABB,AAGG),3个二倍体种(AA,A~uA~u)及B组的可能供体沙融山羊草(Ae. shronensis)体细胞核型和N—带进行了分析。结果表明,小麦属全部为具中部或次中部着丝点染色体,核型属于“2A”类型,不对称性随倍性提高而有所增加。种问核型有一定差异。所有小麦B染色体组、G染色体组和4A染色体均显N—带,其它染色体则不显带或只显很浅的着丝点带。六倍体种B染色体组带型基本相同,四倍体小麦B组N—带种间有一定差异。提莫菲维小麦(T.Timopheevi)G组带纹数目和分布与B梁色体组有显著差别,作者认为两者非同源。沙融山羊草核型和带型都与小麦B组相近,是B组的可能供体。一粒系小麦A染色体组基本不显N—带,其中无与4A带型相同的染色体,4A起源尚待研究。     

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.     

Mammalian chromosomes contain cis-acting elements that control replication timing, mitotic condensation, and stability of entire chromosomes

Recent studies indicate that mammalian chromosomes contain discrete cis-acting loci that control replication timing, mitotic condensation, and stability of entire chromosomes. Disruption of the large non-coding RNA gene ASAR6 results in late replication, an under-condensed appearance during mitosis, and structural instability of human chromosome 6. Similarly, disruption of the mouse Xist gene in adult somatic cells results in a late replication and instability phenotype on the X chromosome. ASAR6 shares many characteristics with Xist, including random mono-allelic expression and asynchronous replication timing. Additional "chromosome engineering" studies indicate that certain chromosome rearrangements affecting many different chromosomes display this abnormal replication and instability phenotype. These observations suggest that all mammalian chromosomes contain "inactivation/stability centers" that control proper replication, condensation, and stability of individual chromosomes. Therefore, mammalian chromosomes contain four types of cis-acting elements, origins, telomeres, centromeres, and "inactivation/stability centers", all functioning to ensure proper replication, condensation, segregation, and stability of individual chromosomes.     

Partial monosomy of long arm of chromosome 4 due to interstitial deletion

Summary A child with congenital malformations and a de novo interstitial deletion of the long arm of chromosome 4 is described. Detailed analysis by G banding revealed the loss of the whole of band q21 and part of bands q13 and q22. The clinical abnormalities are quite dissimilar from those features described in other cases of partial 4q monosomy, which generally appear to result from the deletion of more distally placed segments of the chromosome.     

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。     

Amplicons on human chromosome 11q are located in the early/late-switch regions of replication timing

Amplicons are frequently found in human tumor genomes, but the mechanism of their generation is still poorly understood. We previously measured the replication timing of the genes along the entire length of human chromosomes 11q and 21q and found that many "disease-related" genes are located in timing-transition regions. In this study, further scrutiny of the updated replication-timing map of human chromosome 11q revealed that both amplicons on human chromosomal bands 11q13 and 11q22 are located in the early/late-switch regions of replication timing in two human cell lines (THP-1 and Jurkat). Moreover, examination of synteny in the human and mouse genomes revealed that synteny breakage in both genomes occurred primarily at the early/late-switch regions of replication timing that we had identified. In conclusion, we found that the early/late-switch regions of replication timing coincided with "unstable" regions of the genome.     

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.