Enhanced induction of abnormal telomere FISH signals in response to oxidative DNA damage.

Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we investigated X-ray-induced abnormalities in telomere signals detected by fluorescence in situ hybridization (telomere FISH) in mouse embryo fibroblast cells. The abnormalities were categorized as either extra telomere signals (ETSs) or loss of telomere signals (LTSs). The results indicated that low doses (0.3-0.5 Gy) of X-rays significantly induced ETS but not LTS and that ETS induction was saturated at doses above 0.5 Gy. In addition, treatment with hydrogen peroxide also induced ETS but not LTS. To clarify the involvement of radicals in inducing ETS, we examined the effect of ascorbic acid (AsA) on telomere FISH signals and found that pre-treatment with AsA (5 mM, 2 h), but not post-treatment, significantly suppressed the induction of ETS by X-irradiation. Importantly, neither pre- nor post-treatment with AsA affected X-ray-induced chromosome aberrations. These results suggest that oxidative DNA damage induced by radicals is involved in the induction of ETS. Furthermore, combined treatment with aphidicolin, a DNA replication inhibitor, elevated the induction of ETS by X-irradiation. This observation suggests that DNA replication stress, potentially triggered by oxidative DNA lesions within telomeres, may contribute to the induction of ETS resulting from X-irradiation. Based on these results, we propose that ETS is a sensitive biological marker of oxidative DNA damage in telomere structures.

Monitoring Telomere Maintenance During Regeneration of Annelids.

Telomere shortening is a hallmark of aging and eventually constrains the proliferative capacity of cells. The protocols discussed here are used for monitoring telomeres comprehensively in Aeolosoma viride, a model system for regeneration studies. We present methods for analyzing the activity of telomerase enzyme in regenerating tissue by telomeric repeat amplification protocol (TRAP) assay, for comparing telomere length between existing tissue and newly regenerated tissue by telomere restriction fragment (TRF) assay, as well as for visualizing telomeres by fluorescence in situ hybridization (FISH).

Direct Visualization of DNA Replication at Telomeres Using DNA Fiber Combing Combined with Telomere FISH.

The ability to analyze individual DNA fibers undergoing active DNA synthesis has emerged as a powerful technique in the field of DNA replication. Much of the initial analysis has focused on replication throughout the genome. However, more recent advancements in this technique have allowed for the visualization of replication patterns at distinct loci or regions within the genome. This type of locus-specific resolution will greatly enhance our understanding of the dynamics of DNA replication in regions that provide a challenge to the replication machinery. Here, we describe a protocol that will allow for the visualization of DNA replication through one of the most structurally complex regions in the human genome, the telomeric DNA.

Karyotypes versus Genomes: The Nymphalid Butterflies Melitaea cinxia, Danaus plexippus, and D. chrysippus.

The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary Melitaea cinxia (n = 31), the monarch Danaus plexippus (n = 30), and the African queen D. chrysippus (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG)n type, as found in most insects. M. cinxia and D. plexippus have "conventional" W chromosomes which are heterochromatic in meiotic and somatic cells. In D. chrysippus, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of M. cinxia and D. plexippus. Representation analysis in sequenced female and male D. chrysippus genomes detected an evolutionarily old autosome-Z chromosome fusion in Danaus. Conserved synteny of whole chromosomes, so called "macro synteny", in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in D. chrysippus by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, D. c. chrysippus and D. c. dorippus, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera, Belostoma).

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome-autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X1X2Y/X1X1X2X2), B. elegans (2n = 26 + X1X2Y/X1X1X2X2), B. elongatum (2n = 26 + X1X2Y/X1X1X2X2), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG) n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere-telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.

Cytogenetic Analysis of Telomere Dysfunction.

Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by the removal of telomere-binding proteins are recognized as double-stranded breaks (DSBs). Repair of DSBs is crucial for the maintenance of genome stability. In mammals, DSBs are repaired by either error-prone nonhomologous end joining (NHEJ) or error-free homologous recombination (HR) and can be visualized as chromosomal fusions.

Probing the Telomere Damage Response.

Telomere dysfunctions, rendered through replicative attrition of telomeric DNA or due to the removal of shelterin components, are recognized as DNA double-stranded breaks (DSBs) by the DNA damage repair (DDR) pathway. This leads to the activation of DNA damage checkpoint sensors, including the Mre11-Rad50-Nbs1 (MRN) complex, γ-H2AX and 53BP1, the ATM and ATR signal-transducing kinases, and downstream effectors, including Chk1, Chk2, and p53. Robust DNA damage response signals at dysfunctional telomeres, achieved by the complete deletion of TRF2 or by expressing dominant-negative mutant TPP1ΔRD, can be detected by their association with γ-H2AX and 53BP1 forming "telomere dysfunction induced foci (TIFs)." Induction of TIFs at telomeres provides an opportunity to quantify the extent of telomere dysfunction and monitor downstream signaling pathways.

Monitoring the DNA Damage Response at Dysfunctional Telomeres.

Telomeres are repetitive DNA repeats that cap the ends of all eukaryotic chromosomes. Their proper maintenance is essential for genomic stability and cellular viability. Dysfunctional telomeres could arise through natural attrition of telomeric DNA or due to the removal of shelterin components. These uncapped chromosomal ends are recognized as DSBs by the DDR pathway, leading to the accumulation of DNA damage sensors at telomeres. The association of these DDR proteins with dysfunctional telomeres forms telomere dysfunction induced DNA damage foci (TIFs). Detection of TIFs at telomeres provides an opportunity to quantify the extent of telomere dysfunction and monitor downstream DNA damage signaling pathways. Here we describe a method for the detection of TIFs using a fluorescent in situ hybridization (FISH) approach.