Plasticity of human chromosome 3 during primate evolution

Comparative mapping of more than 100 region-specific clones from human chromosome 3 in Bornean and Sumatran orangutans, siamang gibbon, and Old and New World monkeys allowed us to reconstruct ancestral simian and hominoid chromosomes. A single paracentric inversion derives chromosome 1 of the Old World monkey Presbytis cristata from the simian ancestor. In the New World monkey Callithrix geoffroyi and siamang, the ancestor diverged on multiple chromosomes, through utilizing different breakpoints. One shared and two independent inversions derive Bornean orangutan 2 and human 3, implying that neither Bornean orangutans nor humans have conserved the ancestral chromosome form. The inversions, fissions, and translocations in the five species analyzed involve at least 14 different evolutionary breakpoints along the entire length of human 3; however, particular regions appear to be more susceptible to chromosome reshuffling. The ancestral pericentromeric region has promoted both large-scale and micro-rearrangements. Small segments homologous to human 3q11.2 and 3q21.2 were repositioned intrachromosomally independent of the surrounding markers in the orangutan lineage. Breakage and rearrangement of the human 3p12.3 region were associated with extensive intragenomic duplications at multiple orangutan and gibbon subtelomeric sites. We propose that new chromosomes and genomes arise through large-scale rearrangements of evolutionarily conserved genomic building blocks and additional duplication, amplification, and/or repositioning of inherently unstable smaller DNA segments contained within them.     

Mapping homology between human and black and white colobine monkey chromosomes by fluorescent in situ hybridization

We used in situ hybridization of chromosome specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the white and black colobus (Colobus guereza 2n = 44). The 24 human paints gave 31 signals on the autosomes (haploid male chromosome set). Robertsonian translocations between chromosomes homologus to human 14 and 15, 21 and 22, form colobine chromosomes 6 and 16, respectively. Reciprocal translocations were found between human chromosomes 1 and 10, 1 and 17, as well as 3 and 19. The alternating hybridization signals between human 3 and 19 on Colobus chromosome 12 show that in this case a reciprocal translocation was followed by a pericentric inversion. The hybridization data show that in spite of the same diploid number and similar Fundamental Numbers, the black and white colobine monkey differs from Presbytis cristata, an Asian colobine, by 6 reciprocal translocations. Comparisons with the hybridization patterns in other primates show that some Asian colobines have a more derived karyotype with respect to African colobines, macaques, great apes, and humans. Chromosome painting also clearly shows that similarities in diploid number and chromosome morphology both between colobines and gibbons are due to convergence. Am. J. Primatol. 42:289–298, 1997. © 1997 Wiley-Liss, Inc.     

The mystery of chromosomal translocations in cancer

Chromosomal translocations in human cancer may result in products that can be suppressed by targeting drugs. An example is bcr-abl tyrosine kinase in chronic myelogenous leukemia that can be treated with imatinib mesylate. However, the mechanisms of translocations or exchanges of chromosomal segments are virtually unknown. In this summary, chromosomal translocations in human cancer are compared with 'crossing over' of chromosomal segments occurring during the first meiotic division. Several proposed mechanisms of the exchange of DNA between and among chromosomes are discussed. The conditions that appear essential for these events to occur are listed. Among them are proximity of the involved DNA segments, mechanisms of excising the target DNA, its transport to the new location, and integration into the pre-existing chromosome. The conclusion based on extensive review of the literature is that practically nothing is known about the mechanism of 'crossing over' or translocation. Based on prior work on normal human cells, it is suggested that only one of the two autosomes participates in these events that may include loss of heterozygozity, another common abnormality in human cancer.     

Fluorescence in situ hybridization establishes homology between human and silvered leaf monkey chromosomes,reveals reciprocal translocations between chromosomes homologous to human Y/5, 1/9, and 6/16, and delineates an X1X2Y1Y2/X1X1X2X2 sex-chromosome system

We employed in situ hybridization of chromosome-specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the silvered lead monkey karyotypes (Presbytis cristata 2n=44). The 24 human paints gave 30 signals on the haploid female chromosome set and 34 signals on the haploid male chromosome set. This difference is due to a reciprocal translocation between the Y and an autosome homologous to human chromosome 5. This Y/autosome reciprocal translocation which is unique among catarrhine primates has produced a X₁X₂Y₁Y₂/X₁X₁X₂X₂ sex-chromosome system. Although most human syntenic groups have been maintained in the silvered leaf monkey chromosomes homologous to human chromosomes 14 and 15, 21 and 22 have experienced Robertsonian fusions. Further, the multiple FISH signals provided by libraries to human chromosomes 1/9, 6/16 indicate that these chromosomes have been split by reciprocal translocations. G-banding analysis shows three different forms of chromosome 1 (X₂) which differ by a complex series of inversions in the 10 individuals karyotyped. Comparisons with the hybridization patterns in hylobatids (gibbons and siamang) demonstrate that resemblances in chromosomal morphology and banding previously taken to indicate a special phylogenetic relationship between gibbons and colobines are due to convergence. A. J. Phys. Anthropol. 102:315–327, 1997. © 1997 Wiley-Liss, Inc.     

利用染色体涂染方法建立人和白眉长臂猿(Hylobates hoolock)的比较染色体图谱

除人Y染色体外,本文采用生物素标记的人全部整条染色体特异探针与白眉长臂猿(Hylobates hoolock)有丝分裂中期分裂相进行染色体原位杂交即染色体涂染法以研究人和白眉长臂猿染色体之间的同源性。在白眉长臂猿18对常染色体上检测出了与人22对常染色体同源的59对染色体片段,确定了人和白眉长臂猿之间的精度较高的染色体连锁群。结果表明:自人与白眉长臂猿的祖先分歧以来,大量的染色体间重排(至少发生了39次易位)和染色体内的重排导致了二者核型的差异。根据杂交结果绘制了首份人和白眉长臂猿比较染色体图谱,并结合已有的人和白掌长臂猿(Hylobates lar)(2n=44)和合趾长臂猿(Hylobates syndactylus)(2n=50)的比较染色体图谱对长臂猿属的染色体进化作了初步的探讨。     

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.     

Satellited Y chromosomes: Structure,origin, and clinical significance

Summary Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunction and/or to translocations of other chromosomes is discussed.     

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.     

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.     

Chromosome painting reveals that galagos have highly derived karyotypes

The differences in chromosome number between Otolemur crassicaudatus (2n = 62) and Galago moholi (2n = 38) are dramatic. However, the total number of signals given by hybridizing human chromosome paints to galago metaphases is similar: 42 in O. crassicaudatus and 38 G. moholi. Many human chromosome homologs are found fragmented in each species, and numerous translocations have resulted in chromosomal syntenies or hybridization associations which differ from those found in humans. Only 7 human autosomes showed conserved synteny in O. crassicaudatus, and 9 in G. moholi. Both galago species have numerous associations or syntenies not found in humans: O. crassicaudatus has 11, and G. moholi has 21. The phylogenetic line leading to the last common ancestor of the two galago species accumulated 6 synapomorphic fissions and 5 synapomorphic fusions. Since the divergence of the two galago species, 10 Robertsonian translocations have further transformed the G. moholi karyotype, and 2 fissions have been incorporated into the O. crassicaudatus karyotype. Comparison with other primates, tree shrews, and other mammals shows that both galagos have karyotypes which are a mixture of derived and conserved chromosomes, and neither has a karyotype close to that of the proposed ancestor of all primates. Am J Phys Anthropol 117:319-326, 2002. Published 2002 Wiley-Liss, Inc.     

Isolation and characterization of cloned human DNA fragments carrying reiterated sequences common to both autosomes and the X chromosome.

Several recombinants were identified and purified from a cloned library of human DNA by virtue of their homology to DNA from a mouse-human hybrid cell line containing a single human chromosome, the X, and their lack of homology to mouse DNA. Three recombinants were characterized in detail, and all were homologous to reiterated DNA from the human X chromosome. These recombinants also were homologous to reiterated sequences on one or more human autosomes and, therefore, were not X chromosome specific. The recombinant DNA fragments homologous to human reiterated X DNA were the same fragments homologous to human reiterated autosomal DNA. Digestion of genomic DNAs with several restriction enzymes revealed that the pattern of fragments homologous to one recombinant, lambda Hb2, was the same on autosomes as on the X chromosome, suggesting that the molecular organization of these elements on the X is not distinct from their organization on autosomes.     

Evolution of a human Y chromosome-specific repeated sequence

The structure and evolution of a repetitive sequence on the human Y chromosome has been studied by restriction enzyme analysis of both total DNA and the isolated sequence. The sequence is shown to cross-hybridize to sequences in female DNA forming unstable duplexes. Mouse/human cell hybrids have been used to investigate the pattern of sequence homology on the X chromosome and some autosomes. We conclude that this sequence is related to human satellite III, but shows considerable differences in structure.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review

Sperm chromosome analysis offers the opportunity to gather information about the origin of chromosome aberrations in human germ cells. Over the last 20 years more than 20 000 sperm chromosome complements from normal donors and almost 6000 spermatozoa from men with constitutional chromosome aberrations (inversions, translocations) have been analyzed for structural and numerical chromosome abnormalities, as well as for segregation of the constitutional chromosome aberrations after the sperm had penetrated hamster oocytes. On the other hand, it took only 6 years to screen more than 3 million mature spermatozoa from healthy probands for disomy rates of 20 autosomes (chromosomes 19 and 22 not evaluated) and the sex chromosomes, and for diploidy rates by in situ hybridization techniques. In the present paper the results arising from both methods are compiled and compared. Received: 29 January 1997 / Accepted: 5 March 1997     

The X chromosome and the rate of deleterious mutations in humans

Monosomy for the X chromosome in humans creates a genetic Achilles' heel for nature to deal with. We report that the human X chromosome appears to have one-third the density of the coding sequence of the autosomes and, because of partial shielding from the high mutation rate of the male sex, that it should also have a lower mutation rate than the autosomes (i.e.,.73). Hence, the X chromosome should contribute one quarter (.33x.73=.24) of the deleterious mutations expected from its DNA content. In this way, selection has possibly moderated risks from mutation in X-linked genes that are thought to have been fixed in their syntenic state since the onset of the mammalian lineage. The unexpected difference in the density of coding sequences indicates that our recent, hemophilia B-based estimate of the rate of deleterious mutations per zygote should be increased from 1.3 to 4 (1.3x3).     

Human chromosome variation with two Robertsonian translocations

Summary A woman was found to have 42 autosomes due to engagement of both chromosomes 14 in Robertsonian rearrangements, one with a chromosome 21 and the other with a chromosome 22: t(14q21q) and t(14q22q). The two translocations appear monocentric and by silver staining have no rRNA activity. The t(14q21q) translocation is familial and was ascertained through a nephew with Down syndrome, while the origin of the t(14q22q) translocation was not established. In addition to these two translocations, the woman had XX/XXX sex chromosome mosaicism. She has had two recognized pregnancies, each resulting in the birth of a child with one of the two translocations. Both children are phenotypically normal, as is their mother, the first normal liveborn individual identified with two Robertsonian translocations.     

Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, we investigated an autosome‐Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10–15 Mya. We used single molecule real‐time sequencing reads to assemble the D. pseudoobscura dot chromosome, including this Y‐to‐dot translocation. We find that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ～78 Kb and is not repeat‐dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y‐to‐dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, we do not find a consistent reduction in intron sizes across the Y‐to‐dot region. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y‐to‐dot translocation.     

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.     

The loss of a single telomere can result in instability of multiple chromosomes in a human tumor cell line

Spontaneous telomere loss has been proposed as an important mechanism for initiating the chromosome instability commonly found in cancer cells. We have previously shown that spontaneous telomere loss in a human cancer cell line initiates breakage/fusion/bridge (B/F/B) cycles that continue for many cell generations, resulting in DNA amplification and translocations on the chromosome that lost its telomere. We have now extended these studies to determine the effect of the loss of a single telomere on the stability of other chromosomes. Our study showed that telomere acquisition during B/F/B cycles occurred mainly through translocations involving either the nonreciprocal transfer or duplication of the arms of other chromosomes. Telomere acquisition also occurred through small duplications involving the subtelomeric region of the other end of the same chromosome. Although all of these mechanisms stabilized the chromosome that lost its telomere, they differed in their consequences for the stability of the genome as a whole. Telomere acquisition involving nonreciprocal translocations resulted in the loss of a telomere on the donor chromosome, which consequently underwent additional translocations, isochromosome formation, or complete loss. In contrast, telomere acquisition involving duplications stabilized the genome, although the large duplications created substantial allelic imbalances. Thus, the loss of a single telomere can generate a variety of chromosome alterations commonly associated with human cancer, not only on a chromosome that loses its telomere but also on other chromosomes. Factors promoting telomere loss are therefore likely to have an important role in generating the karyotype evolution associated with human cancer.     

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.