Telomeres and mechanisms of Robertsonian fusion

The Robertsonian (Rb) fusion, a chromosome rearrangement involving centric fusion of two acro-(telo)centric chromosomes to form a single metacentric, is one of the most frequent events in mammalian karyotype evolution. Since one of the functions of telomeres is to preserve chromosome integrity, a prerequisite for the formation of Rb fusions should be either telomere loss or telomere inactivation. Possible mechanisms underlying the formation of various types of Rb fusion are discussed here. For example, Rb fusion in wild mice involves complete loss of p-arm telomeres by chromosome breakage within minor satellite sequences. By contrast, interstitial telomeric sites are found in the pericentromeric regions of chromosomes originating from a number of vertebrate species, suggesting the occurrence of Rb-like fusion without loss of telomeres, a possibility consistent with some form of telomere inactivation. Finally, a recent study suggests that telomere shortening induced by the deletion of the telomerase RNA gene in the mouse germ-line leads to telomere loss and high frequencies of Rb fusion in mouse somatic cells. Thus, at least three mechanisms in mammalian cells lead to the formation of Rb fusions. Received: 11 November 1997 / Accepted: 21 December 1997     

Chromosome banding homologies in Swamp and Murrah buffalo

Silver staining of Swamp buffalo (2n = 48) metaphase chromosomes revealed telomeric nucleolus organizer regions (NOR's) located on five pairs of autosomes identified by R-banding as numbers 4 p (submetacentric), 8, 20, 22, and 23 (acrocentrics); interphase nuclei also showed no more than five nucleoli. The Murrah buffalo (2n = 50) was previously reported to have telomeric NOR's located on six pairs, -3 p and 4 p (submetacentric), 8, 21, 23, and 24 (acrocentrics). By comparing the two types of buffalo it was concluded that: all of the chromosomes are similar in banding patterns; chromosome 1 of Swamp results from a telomere-centromere tandem fusion between two chromosomes identified as 4 p and 9, respectively, in the Murrah karyotype, thus accounting for the reduced diploid number of Swamp buffalo; the fusion causes the loss of NOR's on the telomeres of chromosome 4, thus accounting for the reduced number of NOR chromosome pairs of Swamp; the presence of a pale C-band are in the region of junction between chromosome 4 and 9 involved in the fusion suggests that the centromeric region of the later is retained and altered.     

The molecular characterization of maize B chromosome specific AFLPs

INTRODUCTIONB chromosomes (Bs) are also called supernumer-ary chromosomes, accessory chromosomes or extrachromosomes. They are supernumerary to the stan-dard chromosome (A chromosomes) set, which arefound in hundreds of plants and animals. They areoften morphologicaIly distinct from A chromosomes,being sma1ler and more highly heterochromatic inmost cases. B chromosomes are inherited in a non-Mendelian wap They dO not pair with A chromo-somes, and exhibite meiotic and mitotic instabiIit…     

Telomeric repeat organization--a comparative in situ study between man and rodent

Human, hamster, and mouse chromosomes show both similarities and differences in telomeric organization, detectable with a novel version of the PRINS technique. The differences allowed us to investigate the fate of the telomeres on a chromosome from one species when this chromosome was introduced into the cells of another species. For this purpose, we tested telomeres in cell lines of somatic cell hybrids containing human chromosomes on a rodent background, finding that the telomeres on human chromosomes could not be discriminated from the telomeres on rodent chromosomes. All telomeres in the cell lines were much shorter than the telomeres in normal cells. In the mouse-derived cell lines, half of the mouse chromosomes were fused to other mouse chromosomes at the ends of their short arms. At the points of fusion we were generally unable to detect telomeric signals. In these cell lines, we also found a fraction of chromosomes ends with only one telomeric signal. In chromosomes where both ends showed only one signal, the relative orientation of the signals appeared to be nonrandom with respect to sister chromatids.     

Human telomeric 6; 19 translocation chromosome with a tendency to break at the fusion point

The behavior of a translocation chromosome t(6; 19) in the lymphocytes of a mentally retarded woman with other anomalies has been analyzed. The two chromosomes were attached at the telomeres of their short arms without any apparent deletion. The centromere of chromosome 19 was marked by a primary constriction and the site of the centromere of chromosome 6 by a C-band, but no constriction. The translocation chromosome showed two primary constrictions once in 8,800 metaphases, probably resulting from mitotic crossing-over. One or both chromatids of the translocation chromosome were broken at the attachment point with a frequency of 1/733 cells. In addition, the chromosome was often bent at this point and the translocated chromosomes 19 and 6 showed a differential spiralization. In this characteristic as well as the weakness of the fusion point, this chromosome differed from other translocations; the fusion obviously was not as firm as in translocations in general. The broken-off chromosome 6 did not regain a primary constriction, but had the appearance of a large acentric fragment. The segregation of the translocation chromosome and the fragment gave rise to a complicated mosaicism with various levels of ploidy for the fragment lacking a functional centromere. The data are in quantitative agreement with the equilibrium expectations under the assumption that each fragment goes to either pole at random in mitosis and that cells divide at the same rate regardless of ploidy. The high rate of nondisjunction of the fragment showed that the inactivated centromere of the translocation chromosome did not regain its activity when chromosome 19 with the functional centromere became separated from it. — The fragility and the behavior of the translocation chromosome and the production of telomeric associations are briefly discussed.     

Cytological and molecular analysis of centromere misdivision in maize.

B chromosome derivatives suffering from breaks within their centromere were examined cytologically and molecularly. We showed by high resolution FISH that misdivision of the centromere of a univalent chromosome can occur during meiosis. The breaks divide the centromere repeat sequence cluster. A telocentric chromosome formed by misdivision was found to have the addition of telomeric repeats to the broken centromere. A ring chromosome formed after misdivision occurred by fusion of the broken centromere to the telomere. Pulsed-field electrophoresis analyses were performed on the telocentric and ring chromosomes to identify fragments that hybridize to both the telomeric repeat and the B-specific centromeric repeat. We conclude that healing of broken maize centromeres can be achieved through the mechanisms of addition or fusion of telomeric repeat sequences to the broken centromere.     

Telomere dysfunction and chromosome instability

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.     

Telomeric signals in robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy.

In situ hybridization with synthetic plant telomeric sequences resulted in labeling of all broad bean (Vicia faba) chromosomes at their ends only. Telocentric chromosomes derived by fission of the metacentric satellite chromosome of V. faba also showed signals at both of their ends, whereas the ancestral metacentric did not display signals at its primary constriction, the point of fission. As in V. faba, all acrocentric mouse chromosomes were labeled by in situ hybridization with a vertebrate telomeric probe at both ends of each chromatid exclusively. However, different metacentric Robertsonian chromosomes derived by fusion of defined acrocentrics did not show signals at their primary constrictions. The mechanism of Robertsonian rearrangement leading to a pseudoaneuploid increase or decrease in chromosome number therefore cannot consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material. The additional assumption of a subdetectable deletion of telomeric sequences after fusion and amplification of these sequences following fission is necessary to explain the present observations.     

Mapping the distribution of the telomeric sequence (T(2)AG(3))(n) in rock wallabies,Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization. ii. the lateralis complex

The distribution of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) was examined by fluorescence in situ hybridization in the six Petrogale (rock wallabies) taxa of the lateralis complex. As expected, the (T(2)AG(3))(n) sequence was located at the termini of all chromosomes in all taxa. However, the sequence was also present at several nontelomeric (viz., interstitial and centromeric) sites. The signals identified were associated with either ancient rearrangements involved with the formation of the 2n = 22 plesiomorphic macropodine karyotype or more recent rearrangements associated with karyotypes derived from the 2n = 22 karyotype. Interstitial (T(2)AG(3))(n) signals identified on chromosomes 3 and 4 in all six species of the lateralis complex and a large centromeric signal identified on chromosome 7 in the five subspecies/races of P. lateralis appear to be related to the more ancient rearrangements. Subsequent chromosome evolution has seen these signals retained, lost, or amplified in different Petrogale lineages. Within the lateralis complex, in two submetacentric chromosome derived by recent centric fusions, the telomeric sequence was identified at or near the centromere, indicating its retention during the fusion process. In the two taxa where chromosome 3 was rearranged via a recent centromeric transposition to become an acrocentric chromosome, the telomeric signal was located interstitially.     

The terminal DNA structure of mammalian chromosomes.

In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.     

串叶松香草Giemsa C带和染色中心研究

以Giemsa C带技术处理串叶松香草根尖细胞染色体(2n=14),全部着丝点及第5和第7对染色体短臂端部显稳定的C带,第6对染色体长臂有两条明显的居间带,其他居间带小而不稳定(重复率不高)。间期细胞核染色体呈Rable构型,其着丝点一极最多出现20个染色中心。统计分析表明,靠近着丝点的短臂端带区和居间带区异染色质有易与着丝点区异染色质融合的倾向。分裂中期Giemsa C带数目与间期染色中心数目存在数量对应关系。     

Molecular and cytogenetic analysis of the telomeric (TTAGGG)n repetitive sequences in the Nile tilapia,Oreochromis niloticus (Teleostei: Cichlidae)

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Fast Diploidization in Close Mesopolyploid Relatives of Arabidopsis

Mesopolyploid whole-genome duplication (WGD) was revealed in the ancestry of Australian Brassicaceae species with diploid-like chromosome numbers (n = 4 to 6). Multicolor comparative chromosome painting was used to reconstruct complete cytogenetic maps of the cryptic ancient polyploids. Cytogenetic analysis showed that the karyotype of the Australian Camelineae species descended from the eight ancestral chromosomes (n = 8) through allopolyploid WGD followed by the extensive reduction of chromosome number. Nuclear and maternal gene phylogenies corroborated the hybrid origin of the mesotetraploid ancestor and suggest that the hybridization event occurred ~6 to 9 million years ago. The four, five, and six fusion chromosome pairs of the analyzed close relatives of Arabidopsis thaliana represent complex mosaics of duplicated ancestral genomic blocks reshuffled by numerous chromosome rearrangements. Unequal reciprocal translocations with or without preceeding pericentric inversions and purported end-to-end chromosome fusions accompanied by inactivation and/or loss of centromeres are hypothesized to be the main pathways for the observed chromosome number reduction. Our results underline the significance of multiple rounds of WGD in the angiosperm genome evolution and demonstrate that chromosome number per se is not a reliable indicator of ploidy level.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

High-resolution cytogenetic characterization of telomeric associations in ring chromosome 19

High-resolution cytogenetic studies of a normal individual with ring chromosome 19 indicate that, at the late-to-mid prophase level of band resolution, no apparent chromosomal material is missing, and that telomeric fusion/association, not deletion, is the cause of the ring chromosome formation. Sub-band analysis of the telomeric fusion shows thin chromatin filaments between the telomeres of some of the very elongated ring chromosomes, which cannot be resolved by metaphase chromosme analysis. The ring chromosome found in this individual shows evidence of the characteristic instability associated with ring chromosomes, including duplicated segments, double rings, and subsequent loss of the ring resulting in cells with monosomy 19. The lack of phenotypic effect and the unstable ring behavior, unlike previously reported patients with ring 19, support the formation of this ring by telomeric association.     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2

BACKGROUND: Telomeres are required to prevent end-to-end chromosome fusions. End-to-end fusions of metaphase chromosomes are observed in mammalian cells with dysfunctional telomeres due to diminished function of telomere-associated proteins and in cells experiencing extensive attrition of telomeric DNA. However, the molecular nature of these fusions and the mechanism by which they occur have not been elucidated. RESULTS: We document that telomere fusions resulting from inhibition of the telomere-protective factor TRF2 are generated by DNA ligase IV-dependent nonhomologous end joining (NHEJ). NHEJ gives rise to covalent ligation of the C strand of one telomere to the G strand of another. Breakage of the resulting dicentric chromosomes results in nonreciprocal translocations, a hallmark of human cancer. Telomere NHEJ took place before and after DNA replication, and both sister telomeres participated in the reaction. Telomere fusions were accompanied by active degradation of the 3' telomeric overhangs. CONCLUSIONS: The main threat to dysfunctional mammalian telomeres is degradation of the 3' overhang and subsequent telomere end-joining by DNA ligase IV. The involvement of NHEJ in telomere fusions is paradoxical since the NHEJ factors Ku70/80 and DNA-PKcs are present at telomeres and protect chromosome ends from fusion.     

Distribution of Telomeric DNA Sequences on the X-Radiation-Induced Chromosome Fragments Observed in the Genome of Androgenetic Brook Trout ( Salvelinus fontinalis , Mitchill 1814)

Cytogenetic screening of the androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814) offspring hatched from eggs exposed to 420 Gy of X-radiation before insemination exhibited residues of the irradiated maternal nuclear genome in the form of small chromosome fragments. Remnants of the irradiated chromosomes had different sizes, and their number varied intraindividually from 1 to 15. To efficiently pass through the series of the cell divisions, such chromosome fragments must have had functional kinetochores. Distribution patterns of the telomeric hybridization signals on the chromosome fragments enabled us to distinguish their 3 groups: (i) telomere-less ring chromosomes with fused broken chromosome arms, (ii) rings formed in the course of fusion of the radiation-broken chromosome arm with the opposite telomeric region and exhibiting interstitial telomeric signals at the fusion point, and (iii) chromosome fragments with fused unprotected sister chromatids of 1 broken arm and intact telomeres from the other arm. Disturbances during segregation of such fragments, mainly breakages during anaphase, may partially explain intraindividual variation in the number and size of the chromosome fragments observed in the androgenetic brook trout.     

Association of telomeric regions of salivary gland chromosomes in Drosophila species of the virilis group]

The frequency of participating salivary gland chromosomes in telomeric associations and specific combinations of associated chromosomes were studied in the following species belonging to "virilis" group of Drosophila: D. virilis, D. texana, D. littoralis Sokolov and D. imeretensis Sokolov. In all species studied predominantly telomeres of two chromosomes form an association. In females of the species mentioned the X-chromosome takes part in association twice as frequent as in males. The total length of the chromosome and the presence of subterminal inversions may sometimes influence the frequency of participating the chromosome in associations. However, the data obtained enable to postulate that internal homology of telomeric regions plays the main role in the process. The equal frequency of participating in telomeric associations for definite chromosomes in species studied may resemble the phylogenetic relation of the species.