Structural Organization of Multiple Alphoid Subsets Coexisting on Human Chromosomes 1, 4, 5, 7, 9, 15, 18, and 19

FISH experiments on metaphase chromosomes, interphase nuclei, and extended chromatin were performed to investigate the structural organization of alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Results indicate that multiple subsets present on chromosomes 5, 7, 15, 18, and 19 are organized in structurally distinct and contiguous domains, while those on chromosomes 4 and 9 give perfectly overlapping signals. Chromosome 1 shows a peculiar organization: probe pAL1, specific for this chromosome, detects two distinct domains separated by the subset identified by probe pZ5.1. The order along the chromosome of alphoid subsets lying on chromosomes 5, 7, 15, 18, and 19, organized in distinct blocks, has also been established. The relationship between the structural organization of these alphoid sequences and their evolutionary history in great apes is discussed.     

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.     

Human (Homo sapiens) and chimpanzee (Pan troglodytes) share similar ancestral centromeric alpha satellite DNA sequences but other fractions of heterochromatin differ considerably

The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. © 1995 Wiley-Liss, Inc.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis and Drosophila kanekoi (Diptera: Drosophilidae)

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3S FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.     

Evolutionary breakpoint analysis on Y chromosomes of higher primates provides insight into human Y evolution

Comparative FISH mapping of PAC clones covering almost 3 Mb of the human AZFa region in Yq11.21 to metaphases of human and great apes unravels breakpoints that were involved in species-specific Y chromosome evolution. An astonishing clustering of evolutionary breakpoints was detected in the very proximal region on the long arm of the human Y chromosome in Yq11.21. These breakpoints were involved in deletions, one specific for the human and another for the orang-utan Y chromosome, in a duplicative translocation/transposition specific for bonobo and chimpanzee Y chromosomes and in a pericentric inversion specific for the gorilla Y chromosome. In addition, our comparative results allow the deduction of a model for the human Y chromosome evolution.     

Comparative FISH analysis of distribution of B chromosome repetitive DNA in A an d B chromosomes in two subspecies of Podisma sapporensis (Orthoptera, Acrididae)

FISH analysis of B chromosome repetitive DNA distribution in A and B chromosomes of two subspecies of Podisma sapporensis (P. s. sapporensis and P. s. krylonensis) was performed. In the B chromosomes, C-positive regions contained homologous DNA repeats present also in some C-positive A chromosome regions. Most C-negative regions contained DNA repeats characteristic of A chromosome euchromatic regions. The two subspecies analyzed differed in the location of A chromosome regions enriched with repeats homologous to repeats of B chromosomes. The only common region enriched with these B chromosome repeats in both subspecies was the X chromosome pericentromeric region. The origin of B chromosomes in P. sapporensis is discussed.     

Comparative mapping of human claudin-1 (CLDN1) in great apes

The gene encoding claudin-1 (CLDN1) has been mapped to human chromosome 3 (HSA3; 3q28-->q29) using a radiation hybrid panel. Employing fluorescence in situ hybridization (FISH) we here show that a human P1-derived artificial chromosome (PAC) containing CLDN1 detects the orthologous sites in chromosomes of the great apes, chimpanzee, gorilla, and orangutan. Furthermore, the chromosomal position of CLDN1 was determined in mouse chromosomes by FISH. The position of fluorescent signals is confined to a single chromosomal site in both great apes and mouse and in each case maps to the chromosomal region that has conserved synteny with HSA3 (PTR2q28, GGO2q28, PPY2q38 and MMU16B1). Using a gene-specific probe our results are consistent with reports of the striking similarity of great ape and human genomes as illustrated previously by chromosome painting.     

Cloned fragment of human alphoid DNA--a molecular marker of the pericentromeric region of chromosome 18

From the library of cloned fragments of human DNA we have isolated two recombinant plasmids containing alphoid DNA sequences pBRHS13, pBRHS65. Both cloned sequences hybridized in situ predominantly to pericentromeric regions of chromosome 18 and with less intensity to pericentromeric regions of chromosomes 2, 9, 20, and were characterized by populational copy number polymorphism in homologous chromosomes. These sequences may appear very useful in the diagnostics and cytogenetic analysis of chromosomal aberrations and in studies of polymorphisms of heterochromatic regions of human chromosomes.     

A study of cryptic terminal chromosome rearrangements in recurrent miscarriage couples detects unsuspected acrocentric pericentromeric abnormalities

Fifty chromosomally normal couples with three or more miscarriages were examined using fluorescent in situ hybridisation (FISH) and a library of subtelomere-specific probes together with alphoid repeats mapping to the acrocentric centromeres. Six abnormalities were found. Firstly, a cryptic reciprocal subtelomere translocation between the long arm of a chromosome 3 and the short arm of a chromosome 10. The other five cryptic abnormalities involved the acrocentric chromosome pericentromeric regions and in one case also Yp. Two patients had a rearranged chromosome 13, where the centromeric region was found to be derived from the short arm, centromere and proximal long arm of chromosome 15. Another two patients had a derived chromosome 22, where the centromere was replaced by two other centromeres, one derived from chromosome 14 and the other from either chromosome 13 or 21, while one patient had the subtelomere region of Yp translocated onto the short arm of a chromosome 21. These abnormalities may be the underlying cause of the recurrent miscarriages, because they may result in abnormal pairing configurations at meiosis leading to non-disjunction of whole chromosomes at metaphase I. The frequency of rearrangements seen in the recurrent miscarriage patient population was significantly different from that in the control group ( P=0.0096, Fisher's exact test) due to the acrocentric pericentromeric abnormalities.     

Comparative mapping of human alphoid satellite DNA repeat sequences in the great apes

Heterochromatic regions of chromosomes contain highly repetitive, tandemly arranged DNA sequences that undergo very rapid variation compared to unique DNA sequences that are predominantly conserved. In this study the chromosomal basis of speciation has been looked at in terms of repeat sequences. We have hybridized twenty-one chromosome-specific human alphoid satellite DNA probes to metaphase spreads of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) to investigate the evolutionary relationship of heterochromatic regions among such hominoid species. The majority of the probes did not hybridize to their corresponding equivalent chromosome but presented hybridization signals on non-corresponding chromosomes. Such observations suggest that rapid changes may have occurred in the ancestral alphoid satellite DNA sequence, resulting in divergence among the great ape species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative cytogenetics of human chromosome 3q21.3 reveals a hot spot for ectopic recombination in hominoid evolution

Fluorescence in situ hybridization mapping of fully integrated human BAC clones to primate chromosomes, combined with precise breakpoint localization by PCR analysis of flow-sorted chromosomes, was used to analyze the evolutionary rearrangements of the human 3q21.3-syntenic region in orangutan, siamang gibbon, and silvered-leaf monkey. Three independent evolutionary breakpoints were localized within a 230-kb segment contained in BACs RP11-93K22 and RP11-77P16. Approximately 200 kb of the human 3q21.3 sequence was not present on the homologous orangutan, siamang, and Old World monkey chromosomes, suggesting a genomic DNA insertion into the breakpoint region in the lineage leading to humans and African great apes. The breakpoints in the orangutan and siamang genomes were narrowed down to 12- and 20-kb DNA segments, respectively, which are enriched with endogenous retrovirus long terminal repeats and other repetitive elements. The inserted DNA segment represents part of an ancestral duplication. Paralogous sequence blocks were found at human 3q21, approximately 4 Mb proximal to the evolutionary breakpoint cluster region; at human 3p12.3, which contains an independent orangutan-specific breakpoint; and at the subtelomeric and pericentromeric regions of multiple human and orangutan chromosomes. The evolutionary breakpoint regions between human chromosome 3 and orangutan 2 as well their paralogous segments in the human genome coincide with breaks of chromosomal synteny in the mouse, rat, and/or chicken genomes. Collectively our data reveal reuse of the same short recombinogenic DNA segments in primate and vertebrate evolution, supporting a nonrandom breakage model of genome evolution.     

Analysis of pericentromeric chromosome 21 specific YAC clones by FISH: Identification of new markers for molecular-cytogenetic application

Fluorescence in situ hybridization (FISH) of chromosome 21 specific yeast artificial chromosome (YAC) clones after Alu-PCR (polymerase chain reaction) amplification has been used to find new region-specific DNA probes for the heterochromatic region of chromosome 21. Six overlapping YAC clones from a pericentromeric contig map (region 21cen-21q11) were analyzed. Four YAC clones were characterized as hybridizing to several chromosomal locations. They are, therefore, either chimeric or shared by different chromosomes. Two of them containing alphoid satellite DNA, are localized at the centromeric regions of chromosomes 13 and 21 (clone 243A11), and on 13cen, 21cen and 1q3 (clone 781G5); the two others are localized at both 21q11 and 13q2 (clone 759D3), and at 18p (clone 770B3). Two YACs were strongly specific for chromosome 21q11 only (clones 124A7 and 881D2). These YACs were used effectively as probes for identifications of chromosome 21 during metaphase and interphase analysis of 12 individuals, including three families with Down syndrome offspring, and 6 amniocyte samples. The location of YAC clones on 21q11 close to the centromeric region allows the application of these clones as molecular probes for the analysis of marker chromosomes with partial deletions of the long arm as well as for pre- and postnatal diagnosis of trisomy 21 when alphoid or more distal region-specific DNA probes are uninformative. Overlapping YAC clones covering human chromosome 21q may be systematically used to detect a set of band-specific DNA probes for molecular-cytogenetic application.     

The evolution of the azoospermia factor region AZFa in higher primates

Clones of a PAC contig encompassing the human AZFa region in Yq11.21 were comparatively FISH mapped to great ape Y chromosomes. While the orthologous AZFa locus in the chimpanzee, the bonobo and the gorilla maps to the long arm of their Y chromosomes in Yq12.1-->q12.2, Yq13.1-->q13.2 and Yq11.2, respectively, it is found on the short arm of the orang-utan subspecies of Borneo and Sumatra, in Yp12.3 and Yp13.2, respectively. Regarding the order of PAC clones and genes within the AZFa region, no differences could be detected between apes and man, indicating a strong evolutionary stability of this non-recombining region.     

自身基因组原位杂交揭示植物基因组重复DNA沿染色体的分布

重复DNA沿染色体的分布是认识植物基因组的组织和进化的要素之一。本研究采用一种改良的基因组原位杂交程序,对基因组大小和重复DNA数量不同的6种植物进行了自身基因组原位杂交(self-genomic in situ hybridization,self-GISH)。在所有供试物种的染色体都观察到荧光标记探针DNA的不均匀分布。杂交信号图型在物种间有明显的差异,并与基因组的大小相关。小基因组拟南芥的染色体几乎只有近着丝粒区和核仁组织区被标记。基因组相对较小的水稻、高粱、甘蓝的杂交信号分散分布在染色体的全长,但在近着丝粒区或近端区以及某些异染色质臂的分布明显占优势。大基因组的玉米和大麦的所有染色体都被密集地标记,并在染色体全长显示出强标记区与弱标记或不标记区的交替排列。此外,甘蓝染色体的所有近着丝粒区和核仁组织区、大麦染色体的所有近着丝粒区和某些臂中间区还显示了增强的信号带。大麦增强的信号带带型与其N-带带型一致。水稻自身基因组原位杂交图型与水稻Cot-1DNA在水稻染色体上的荧光原位杂交图型基本一致。研究结果表明,自身基因组原位杂交信号实际上反映了基因组重复DNA序列对染色体的杂交,因而自身基因组原位杂交技术是显示植物基因组中重复DNA聚集区在染色体上的分布以及与重复DNA相关联的染色质分化的有效方法。     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.