The evolutionary origin of human subtelomeric homologies--or where the ends begin

The subtelomeric regions of human chromosomes are comprised of sequence homologies shared between distinct subsets of chromosomes. In the course of developing a set of unique human telomere clones, we identified many clones containing such shared homologies, characterized by the presence of cross-hybridization signals on one or more telomeres in a fluorescence in situ hybridization (FISH) assay. We studied the evolutionary origin of seven subtelomeric clones by performing comparative FISH analysis on a primate panel that included great apes and Old World monkeys. All clones tested showed a single hybridization site in Old World monkeys that corresponded to one of the orthologous human sites, thus indicating the ancestral origin. The timing of the duplication events varied among the subtelomeric regions, from approximately 5 to approximately 25 million years ago. To examine the origin of and mechanism for one of these subtelomeric duplications, we compared the sequence derived from human 2q13--an ancestral fusion site of two great ape telomeric regions--with its paralogous subtelomeric sequences at 9p and 22q. These paralogous regions share large continuous homologies and contain three genes: RABL2B, forkhead box D4, and COBW-like. Our results provide further evidence for subtelomeric-mediated genomic duplication and demonstrate that these segmental duplications are most likely the result of ancestral unbalanced translocations that have been fixed in the genome during recent primate evolution.     

An interspersed repeated sequence specific for human subtelomeric regions.

A family of DNA loci (DNF28) from the pseudoautosomal region of the human sex chromosomes is characterized by a repeated element (STIR: subtelomeric interspersed repeat) which detects homologous sequences in the telomeric regions of human autosomes by in situ hybridization. Several STIR elements from both the pseudoautosomal region and terminal parts of autosomes were cloned and sequenced. A conserved 350 bp sequence and some characteristic structural differences between the autosomal and pseudoautosomal STIRs were observed. Screening of the DNA sequence databases with a consensus sequence revealed the presence of STIRs in several human loci localized in the terminal parts of different chromosomes. We mapped single copy probes flanking the cloned autosomal STIRs to the subtelomeric parts of six different chromosomes by in situ hybridization and genetic linkage analysis. The linkage data show a greatly increased recombination frequency in the subtelomeric regions of the chromosomes, especially in male meiosis. The STIR elements, specifically located in subtelomeric regions, could play a role in the peculiar recombination properties of these chromosomal regions, e.g. by promoting initiation of pairing at meiosis.     

Personalized evolutionary hypothesis of breast neoplasm based on differential hybridization signals of subtelomeric probes

The limited availability of data on somatic gene evolution in breast cancer is challenging item to apply an appropriate clinical management for each individual. As human subtelomeric sequences are diverse‐prone and variable, they can be considered as hot spots for analysis of the health or disease status. We compared the hybridization signals of subtelomeric sequences in normal cells with those in auxiliary lymph nodes (ALNs) isolated from a single patient. Distinct signal intensities were found in all chromosomes: weak (5, 6, 9–12, and 16–19), medium (1, 5–9, 19, and X), and strong (2, 5, 9, 10, 16, and 18) intensities. Signal intensities in the patient's ALN and lymphocytes were higher than in normal tissues. The absence and presence of one or more hybridization signals and the presence of signals in the p and q arms were also variable. Whereas chromosomes 2, 3, 5, 7, 8, 10, 16, 18, 20, and X showed three hybridization spots, chromosomes 1, 4, 9, 12, 17, and 18–19 presented a reduced signal in the ALN and lymphocytes. In addition, signal intensity in the p arm was higher than in the q arm in most patients’ chromosomes. Therefore, we propose that subtelomeric hybridization be followed periodically in individuals with breast neoplasm to provide specific patterns. Such profiling could be considered as a prediction marker throughout the patients’ life. Together with the Ki67, cyclin D1, and cyclin E expression profiles, the subtelomeric hybridization profile could provide complementary information for cancer management.     

Isolation of a cancer-associated microchromosome in the sperm-dependent parthenogen Poecilia formosa

In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence.     

Application of mFISH for the analysis of chemically-induced chromosomal aberrations: a model for the formation of triradial chromosomes

Using a human lymphoblastoid cell line WTK-1, we applied multicolor fluorescence in situ hybridization (mFISH) technique to analyze mitomycin C (MMC)-induced chromatid exchanges, focusing especially on the triradial chromosomes. It was found that the triradial chromosomes were formed with a specific rearrangement, "recipient and donor" relationship. The exchange sites of the recipient chromosomes were on single chromatid breaks and distributed randomly throughout the interstitial, pericentromeric, and terminal regions. In counterpart, donor chromosomes exchanged on isochromatid breaks of their telomeric and/or subtelomeric regions with the single chromatid breaks of recipient chromosomes. More than 80% of the scored triradial chromosomes were formed with such rearrangements, and few acentric chromosome fragments derived from the donor chromosomes could be detected in the metaphases observed. We therefore suggest that biological mechanisms of breakages between the recipient and donor chromosomes are different: the former due to direct DNA-damage by MMC, but the latter due to indirect DNA-damage depending on telomeric specific structure/function.     

The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes.

PfEMP1, a Plasmodium falciparum-encoded protein on the surface of infected erythrocytes is a ligand that mediates binding to receptors on endothelial cells. The PfEMP1 protein, which is encoded by the large var gene family, shows antigenic variation and changes in binding phenotype associated with alterations in antigenicity. We have constructed a yeast artificial chromosome contig of chromosome 12 from P. falciparum and show that var genes are arranged in four clusters; two lie amongst repetitive subtelomeric sequences and two occur in the more conserved central region. Analysis of parasite chromosomes by pulsed field gel electrophoresis (PFGE) demonstrates that most contain var genes and two-dimensional PFGE has shown that var genes are located at chromosome ends interspersed amongst repetitive sequences present in the subtelomeric complex. Analysis of a var gene located in the subtelomeric region of chromosome 12 has shown that it has close homologues at the opposite end of the chromosome and in the subtelomeric region of two other chromosomes. This suggests that recombination between heterologous chromosomes has occurred in the subtelomeric regions of these chromosomes. The subtelomeric location of var genes dispersed amongst repetitive sequences has important implications for generation of antigenic variants and novel cytoadherent specificities of this protein.     

In situ localization of human fibronectin (FN) genes to chromosome regions 2p14----p16, 2q34----q36, and 11q12.1----q13.5 in germ line cells, but to chromosome 2 sites only in somatic cells

The locations of the genes for fibronectin (FN) on chromosomes of human germ line and somatic cells were determined by in situ molecular hybridization with two 3H-labeled DNA probes, one for the region encoding the cell attachment domain of human FN, the other for the 3' noncoding and part of the coding region. Pachytene chromosomes of two males and lymphocyte chromosomes of one of these males and a female were used. Two regions of hybridization on pachytene and somatic chromosome 2 (p14----p16 and q34----q36) were found, but not in all individuals. A third region of hybridization was found at 11q12.1----q13.5 in meiotic, but not with significant frequency in somatic chromosomes. It is not clear if these differences between meiotic and somatic chromosomes, and the large differences between individuals at some of the other hybridization sites, resulted solely from technical factors. The differences between the findings in meiotic and somatic preparations might be due to the presence of four strands in pachytene chromosomes versus only one per somatic chromatid. Individual differences in DNA sequences in the chromosome segment containing the gene, differences in gene locations among individuals, or between meiotic and mitotic chromosomes might account for the other findings. The results confirm some of the earlier studies with cell hybrids that mapped FN genes to chromosomes 2 or 11. The combined findings suggest that some of these loci may be coding for the plasma form of FN and others for the cellular form. The expression of the different FN types by differentiated cells might then depend on the loci that are activated.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

Physical Mapping of Human 7q and 14q Subtelomeric DNA Sequences in the Great Apes

Phylogenetic divergence of the members of the Pongidae familyhas been based on genetic evidence. The terminal repeat array(T₂AG₃) has lately been considered as an additional basis toanalyze genomes of highly related species. The recent isolationof subtelomeric DNA probes specific for human (HSA) chromosomes7q and 14q has prompted us to cross-hybridize them to the chromosomesof the chimpanzee (PTR), gorilla (GGO) and orangutan (PPY) tosearch for its equivalent locations in the great ape species.Both probes hybridized to the equivalent telomeric sites ofthe long (q) arms of all three great ape species. Hybridizationsignals to the 7q subtelomeric DNA sequence probe were observedat the telomeres of HSA 7q, PTR 6q, GGO 6q and PPY 10q, whilehybridization signals to the 14q subtelomeric DNA sequence probewere observed at the telomeres of HSA 14q, PTR 15q, GGO 18qand PPY 15q. No hybridization signals to the chromosome 7-specificalpha satellite DNA probe on the centromeric regions of theape chromosomes were observed. Our observations demonstratesequence homology of the subtelomeric repeat families D7S427and D14S308 in the ape chromosomes. An analogous number of subtelomericrepeat units exists in these chromosomes and has been preservedthrough the course of differentiation of the hominoid species.Our investigation also suggests a difference in the number ofalpha satellite DNA repeat units in the equivalent ape chromosomes,possibly derived from interchromosomal transfers and subsequentamplification of ancestral alpha satellite sequences.     

Replication Timing of Human Telomeres Is Chromosome Arm–Specific,Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

Conservation of human-derived pseudoautosomal sequences on the sex chromosomes of the great apes

In situ hybridization using a repeated element specific for the human pseudoautosomal region, DXYZ2, revealed the presence of this repeat in the early replicating portion of the sex chromosomes of the great apes. This segment, as well as the DXYZ2 repeats, are located in band Xp22.3 and in a telomeric or subtelomeric region of the Y chromosome. These segments may therefore represent pseudoautosomal regions, as in man.     

Subtelomeric rearrangements in Polish subjects with intellectual disability and dysmorphic features

Fluorescent in situ hybridization (FISH) was performed in 76 patients referred to our department because of intellectual disability and dysmorphic features that can be related to subtelomeric microaberrations. In all the patients, conventional cytogenetic methods revealed normal karyotype. Four (5.3%) subtelomeric rearrangements were detected by FISH: 2 subtelomeric 1p36 deletions, an unbalanced translocation involving chromosomes 1 and 12 with 1p36 deletion, and a de novo balanced translocation involving chromosomes 19 and 22. Thus, 3 cases of 1p36 subtelomeric deletion were found (3.95%). To confirm subtelomeric rearrangements in 2 patients, comparative genomic hybridization (CGH) was applied. Moreover, 3 cases of polymorphism without phenotypic effects were found: in 2 patients, the polymorphism involved the long arm of chromosome 2 (maternal derivative in both patients), while in the third patient, a polymorphism of the long arm of chromosome 7 was diagnosed. The latter polymorphism was also found in the patient’s mother and grandfather.     

The mobile nature of acrocentric elements illustrated by three unusual chromosome variants

Variant chromosomes are polymorphic in areas that are rich in repeat sequences such as the pericentromeric regions or in the acrocentric short arm regions. The dynamic nature of these regions is evident in the polymorphisms they exhibit. In this paper three unusual variants are described: a chromosome 21 with additional material on its short arm, a chromosome 7 with an insertion in the short arm and a chromosome 2 with satellites at the end of the long arm. All three variants were shown to involve acrocentric elements using special banding techniques and fluorescence in situ hybridization. The 21 variant was found to be a tricentric with a 21 and two 15 alpha-, two classical and three acrocentric beta-satellite signals interspersed by AgNOR-positive regions. The telomeres were present at the two terminal ends. The insertion on chromosome 7 was found to be C-band positive and to contain acrocentric beta-satellite DNA. However, acrocentric alpha-satellite, classical satellite, whole-chromosome-painting or all-telomeres sequence probes did not hybridize to the insertion. The satellited region of chromosome 2 had two C-bands, a small positive all-centromeres probe signal, and two signals for the beta-satellite probe. Sandwiched between the beta-satellite sequences was an AgNOR-positive region. The telomeres were present at the two ends of the satellited chromosome 2. Chromosome 2 subtelomeric probes hybridized to the terminal ends of the short and long arm of chromosome 2. The common thread in these three variants is the involvement of acrocentric short arm elements. The acrocentric short arm elements are shown to move to other acrocentric or nonacrocentric chromosomes and relocate to both terminal and interstitial positions. The integrations are stable and heritable. Received: 23 September 1997 / Accepted: 23 February 1998     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities

A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach. We used an alternative approach of genomic walking, starting from a subtelomeric sequence, TelBam3.4, present on a number of human chromosomes including 22q. This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q. The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q. The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22. In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis. Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations. Received: 21 November 1995