Fragile site and interstitial telomere repeat sequences at the fusion point of a de novo (Y;13) translocation

We describe a novel fragile site in a rearranged chromosome, associated with the presence of telomeric repeat sequences at the fusion point of a translocation between chromosomes 13 and Y. The case reported in this study shows a de novo (Y;13) translocation, which appears to represent fusion of an apparently intact chromosome Y with a chromosome 13 that has lost only part of its short arm. Ten percent of the cells show a normal karyotype without the (Y;13) translocation. Molecular cytogenetic studies of the derived Y;13 chromosome revealed three hybridization sites of the telomeric probes – one at each end and one at the breakpoint junction. A fragile site is also observed in the intrachromosomic telomeric region. This coincidence suggests that the telomere repeat sequences (TTAGGG)n, when present at an interstitial chromosomal location, can promote the formation of a novel fragile site. Received: 15 November 1995 / Revised: 6 March 1996     

Human intrachromosomal telomeric-like repeats: sequence organization and mechanisms of origin

The intrachromosomal location of (T2AG3)n telomeric sequences has been reported in several species. It was proposed that interstitial telomeres (ITs) originated through telomeric fusion of ancestral chromosomes. However, the data so far obtained derive mainly from cytogenetic observations. Cloning and database searching of human IT sequences allowed us to identify three classes: (i) short ITs, composed of few, essentially exact T2AG3 units; (ii) subtelomeric ITs, composed of larger arrays (several hundred base pairs) including many degenerate units within subtelomeric domains; (iii) fusion ITs, in which two extended stretches of telomeric repeats are oriented head-to-head. The number of short ITs is over 50 and subtelomeric ITs are probably present at all chromosomal ends. Surprisingly, the telomeric sequence in 2q13 remains the only fusion IT so far characterized, and evidence presented here suggests that another member of this class may be present in 1q41. Different molecular mechanisms generated the three classes. In particular, several short ITs interrupt precisely repetitive elements or are flanked by direct repeats of 10-41 bp, and are conserved in gorilla and chimpanzee. These features strongly suggest that telomeric repeats were inserted at intrachromosomal sites through the repair of double-strand breaks that occurred in the germline during evolution.     

Newly identified repeat sequences, derived from human chromosome 21qter, are also localized in the subtelomeric region of particular chromosomes and 2q13, and are conserved in the chimpanzee genome

Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.     

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.     

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley-Liss, Inc.     

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     

Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.     

A sequence-ready map of the human chromosome 1q telomere

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.     

Telomeric fusion in pre-T-cell acute lymphoblastic leukemia

Summary Telomeric fusion, a rare phenomenon, was observed in malignant cells from the peripheral blood of an 18-year-old male with rapidly progressive pre-T-cell acute lymphoblastic leukemia (ALL). Only two comparable cases, both with B-cell ALL, have been reported with telomeric fusion in neoplasia. All of the leukemic cells examined from our patient had two chromosome abnormalities consisting of partial triplication (trp) of chromosome 2 and a derivative chromosome 3. Approximately a third of the leukemic cells showed in addition telomere-telomere fusions. These involved the telomeric regions of 1p, 2p, 4q, 5q, 7q, 10q, 11q, 12p, 15p, 21p, and Xq and 3p of the derivative (3). The findings in this case suggest that telomeric fusion may function as a mechanism for the development of chromosome rearrangements that may play a role, albeit rarely, in human neoplasia.     

Segregation analysis of rare autosomal fragile sites

Summary Segregation analyses were performed on pedigrees with rare autosomal fragile sites. The results of the analysis of pedigrees with folate sensitive fragile sites, including 2q1, 6p23, 7p11, 8q22, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, and 20p11, suggested that expression of the gene depended on the carrier parent: it was only 50% penetrant when transmitted by a carrier father, but fully penetrant when transmitted by a carrier mother. Pedigrees with the bromodeoxyuridine (BrdU) fragile site, fra(10)(q25), showed the same trend but the results were not statistically significant. In addition, 38 of the 44 probands with folate sensitive or BrdU-sensitive fragile sites received the gene from their carrier mother and only six received it from their father. In contrast, the analysis of pedigrees with the distamycin A-inducible site, fra(16)(q22), gave the results expected for a simple codominant trait with complete penetrance. Probands with this fragile site received the gene equally from mothers or fathers. The genetic implications of these results are discussed.     

Evolutionary conservation of fragile sites induced by 5-azacytidine and 5-azadeoxycytidine in man,gorilla, and chimpanzee

Summary Lymphocyte cultures from man, gorilla, and chimpanzee were treated with 5-azacytidine and 5-azadeoxycytidine. These cytidine analogues induce common fragile sites in the chromosome bands 1q42 and 19q13 of man. A rare fragile site is induced by 5-azadeoxycytidine in the band 1q24. The optimum conditions required for inducing these new fragile sites were determined by a series of experiments. The common fragile site in human chromosome 1q42 also exists in the gorilla and chimpanzee in the homologous band 1q32. The fragile site in human chromosome 19q13 was demonstrated in the gorilla in the homologous chromosome band 20q13. These are the first examples found of evolutionary highly conserved fragile sites in homologous chromosome bands in related primate species. The interaction between 5-azacytidine, 5-azadeoxycytidine, and chromosomal DNA; the evolutionary conservation of genes located within or closely adjacent to the fragile sites in the chromosome 1 of Hominoidea; and the phylogenetic origin of the two new common fragile sites are discussed.     

Isolation and characterization of a mouse subtelomeric sequence

A mouse subtelomeric sequence, ST1, was generated from genomic DNA of the mouse HR9 (129/Sv origin) cell line by the polymerase chain reaction (PCR) using a single telomeric primer. ST1 was cloned and characterized: it is composed of 670 bp of novel DNA sequence flanked on each end by inverted telomeric hexanucleotide repeats (TTAGGG)_n. PCR amplification from BALB/c mouse DNA using this single primer gave the same major product. Southern analysis and PCR using internal ST1 primers confirmed that the ST1 sequence is present in mouse genomic DNA. In situ hybridization to metaphase chromosomes of SJL origin mapped ST1 to many, if not every, mouse telomere. PCR experiments using different combinations of the telomeric, minor satellite, and ST1 primers indicated that some ST1 copies are adjacent to minor satellite sequences, that telomeric and ST1 sequences are not generally interspersed with minor satellite sequences,and that ST1 and the minor satellite have a consistent and specific orientation relative to each other and to the telomere.by H.F. Willard     

Organization of subtelomeric repeats in Plasmodium berghei.

Several (but not all) Plasmodium berghei chromosomes bear in the subtelomeric position a cluster of 2.3-kilobase (kb) tandem repeats. The 2.3-kb unit contains 160 base pairs of telomeric sequence. The resulting subtelomeric structure is one in which stretches of telomeric sequences are periodically spaced by a 2.1-kb reiterated sequence. This periodic organization of internal telomeric sequences might be related to chromosome-size polymorphisms involving the loss or addition of subtelomeric 2.3-kb units.     

Chromosome translocation in Plasmodium berghei.

We describe a chromosome translocation in a karyotype mutant of the rodent malarial parasite Plasmodium berghei. In this mutant (named EP) a small chromosome (chromosome 7), which has exhibited a size range between 0.9 and 1.4 Mb in other clones of P. berghei, is translocated to chromosome 13 or 14 with a size of about 3 Mb. By comparison of Apa-I restriction fragments of the chromosomes from mutant EP and from a reference clone (named HP) of P. berghei, we found evidence for a junction of subtelomeric chromosome 7 sequences and internal chromosome 13/14 sequences. In addition, a new chromosome of 1.4 Mb (named EP7) is present in mutant EP, which is (mainly) composed of sequences of chromosome 13/14. EP7 contains one telomeric region derived from chromosome 13/14. We found evidence that internal sequences of chromosome 13/14 are joined to telomeric sequences in the other telomeric region of EP7. The karyotype of mutant EP was stable during asexual and sexual multiplication and we found no indications for phenotypic changes.     

A low-copy repeat located in subtelomeric regions of 14 different human chromosomal termini.

Two cosmid clones, cG1A3 and cG1A6, containing large blocks of a telomeric repeat sequence, (TTAGGG)n, were isolated independently. Restriction mapping and fingerprinting studies suggested that cosmid cG1A6 is entirely contained within cG1A3. Competitive in situ hybridization localized cG1A3 to 14 different human telomeres and one internal site at 2q14----q21, indicating the presence of a subtelomeric low-copy repeat within this cosmid.     

Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

A complex network of interactions governs DNA methylation at telomeric regions

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.     

Genomic organization and chromosomal localization of the mouse protein kinase C alpha gene

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.