Studying the biological and technical sources of variation in telomere length of individual chromosomes.

BACKGROUND: Consistent average length differences between species and chromosome arm differences within species indicate that telomere length is genetically determined. This seems to contradict an observed large variation in lengths of the same human telomere between metaphases of the same individual. We examined the extent to which the variation in the telomeres of the human X and Y chromosomes is heritable, induced, or technical in origin. METHODS: Metaphase chromosomes were stained by fluorescence in situ hybridization with a telomere repeat-specific probe, and fluorescence intensities of the X and Y chromosomes were measured. If telomere length variation is predominantly genetically determined and a 50% probability of meiotic recombination between the pseudo-autosomal regions of Yp and Xp in the father is taken into account, one expects an equal chance that the Yp telomere of a son is derived from his father's Xp or Yp telomere. This implies that the Yp/Yq telomere ratios in fathers and sons will be identical in the absence of paternal meiotic recombination and different when recombination occurs. RESULTS: Among five father-son pairs, four showed similar Yp/Yq ratios (P > 0.05), whereas one pair exhibited a large difference in the Yp/Yq ratio that was attributable to a significantly longer Xp than Yp telomere in the father and a presumptive meiotic exchange between X and Y during paternal meiosis. Further, the Xq telomere exhibited a generally shorter telomere length than the others. CONCLUSIONS: The high variation in telomere length appeared to be intracellular (between sister chromatids) and, hence, technical in nature. We found no measurable induced variation in the cells studied, implying that, if induced variation exists, it is small compared with the technical variation.     

Telomere dynamics and genome stability in the human pancreatic tumor cell line MIAPaCa-2

Telomeres are specialized structures found at the ends of eukaryotic chromosomes serving as guardians of genome stability. In normal cells telomeres shorten with each cell division, but immortal cells undergoing multiple divisions constantly have to maintain telomere lengths above a critical level. This is accomplished either through expression of telomerase or the alternative recombination pathway (ALT). In the present study, we analyzed telomere dynamics of the telomerase positive human pancreatic tumor cell line MIAPaCa-2. The cells demonstrated genomic instability with a high frequency of chromosomal aberrations resulting in differences between individual karyotypes within the same cell population. The telomeres were short when compared with normal human fibroblasts, and about 39% of the chromosome ends did not have detectable telomere repeats as demonstrated by PNA-FISH. In many cases telomere signals were missing even when sister chromatids were strongly labeled. In addition, we used an internal PNA probe specific for the X chromosome, present in a single copy in these cells, in order to follow telomere dynamics on individual chromatids. High heterogeneity in telomere signals among individual X chromosomes as well as between their sister chromatids suggested sudden and stochastic loss or gain of telomere repeats. Such constant genomic instability often results in apoptosis and death of a fraction of cells present in the culture at all times. We discuss possible molecular mechanisms that may explain this observed telomere heterogeneity and possible adaptive repair mechanisms by which these cells maintain their chromosomes in order to survive such extreme and permanent genomic instability.     

Chinese hamster telomeres are comparable in size to mouse telomeres.

We report here the results of a telomere length analysis in four male Chinese hamsters by quantitative fluorescence in situ hybridization (Q-FISH). We were able to measure telomere length of 64 (73%) of 88 Chinese hamster telomeres. We could not measure telomere length in chromosome 10 or in the short arms of chromosomes 5, 6, 7 and 8 because of the overlaps between the interstitial and terminal telomeric signals. Our analysis in the 73% of Chinese hamster telomeres indicate that their average length is approximately 38 kb. Therefore, Chinese hamster telomeres are comparable in length to mouse telomeres, but are much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our results indicate that individual Chinese hamster chromosomes may have specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.     

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.     

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.     

Telomere length measurement in mouse chromosomes by a modified Q-FISH method

Telomeres are physical ends of mammalian chromosomes that dynamically change during the lifetime of a cell or organism. In order to understand mechanisms responsible for telomere dynamics, it is necessary to develop methods for accurate telomere length measurement. The most sensitive method for measuring telomere length in mouse chromosomes is quantitative fluorescence in situ hybridization (Q-FISH). The usual protocol for Q-FISH requires plasmids with variable numbers of telomeric repeats and fluorescence beads as calibration standards. Here, we describe a Q-FISH protocol in which two mouse lymphoma cell lines with well-defined telomere lengths are used as calibration standards. Using this protocol we demonstrate that reproducible results can be obtained in a set of four different mouse cell lines. This method can be adapted so that any pair of mammalian cell lines can serve as an internal calibration standard.     

Mouse satellite DNA, centromere structure, and sister chromatid pairing

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.     

Differential fluorescence of sister chromatids with 4'-6-diamidino-2-phenylindole.

Differential fluorescence of sister chromatids and sister chromatid exchanges (SCE) in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with 4'-6-diamidino-2-phenylindole (DAPI). The DAPI fluorescence appears to be more stable than that of the dye 33258 Hoechst and may provide a more sensitive method for the detection of SCE.     

Endomitosis in the mealy bug,Planococcus citri (Homoptera: Coccoidea)

Endomitosis in the Malpighian tubules of the mealy bug Planococcus citri (Risso) is described. The stages are identified on the basis of the length of the chromosomes and the distance between the sister chromatids or chromosomes. The appearance of the chromosomes in the various stages of endomitosis is compared to that in other hemipteran insects. During anaphase and telophase of endomitosis the ends of the sister chromatids and chromosomes tend to stay together longer than the other parts. It is suggested that in holokinetic chromosomes special regions for holding the chromatids together are concentrated near the ends of the chromosomes.Supported by grant GB1585 from the National Science Foundation, Washington, D.C.     

The relative lengths of individual telomeres are defined in the zygote and strictly maintained during life

Previous studies have indicated that average telomere length is partly inherited ( Slagboom et al., 1994 ; Rufer et al., 1999 ) and that there is an inherited telomere pattern in each cell ( Graakjaer et al., 2003 ); ( Londoño‐Vallejo et al., 2001 ). In this study, we quantify the importance of the initially inherited telomere lengths within cells, in relation to other factors that influence telomere length during life. We have estimated the inheritance by measuring telomere length in monozygotic (MZ) twins using Q‐FISH with a telomere specific peptide nucleic acid (PNA)‐probe. Homologous chromosomes were identified using subtelomeric polymorphic markers. We found that identical homologous telomeres from two aged MZ twins show significantly less differences in relative telomere length than when comparing the two homologues within one individual. This result means that towards the end of life, individual telomeres retain the characteristic relative length they had at the outset of life and that any length alteration during the lifespan impacts equally on genetically identical homologues. As the result applies across independent individuals, we conclude that, at least in lymphocytes, epigenetic/environmental effects on relative telomere length are relatively minor during life.     

Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes]

The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C-negative regions, as well as between G-bands and R-bands, has been produced.     

Telomere Length Correlations among Somatic Tissues in Adult Zebra Finches

Telomeres are repetitive non coding DNA sequences located at the end of eukaryotic chromosomes, which maintain the integrity of the genome by hiding the chromosome ends from being recognised as double stranded breaks. Telomeres are emerging as biomarkers for ageing and survival, and are susceptible to reflect different individual life history trajectories. In particular, the telomere length with which one starts in life has been shown to be linked with individual life-long survival, suggesting that telomere dynamics can be a proxy for individual fitness and thereby be implicated in evolutionary trade-offs. As a consequence, an increasing number of studies were conducted on telomeres in the fields of ecology and evolutionary biology, in which telomere length was almost exclusively measured from blood samples. However, not only do the number of repeats of the telomeric sequences vary among species, but also within species with great inter-individual telomere lengths variability with age, tissues, and chromosomes. This raises the issue of the exact biological meaning of telomere measurement in blood cells and stimulated the study of the correlation of telomere lengths among tissues over age. By measuring telomere length in adult zebra finches (Taeniopygia guttata) in different somatic tissues displaying variable cell turnovers (bone marrow, brain, spleen, pectoral muscle, heart, liver and in red blood cells), we checked that the measure of telomere length in red blood cells is related to telomere lengths in the other tissues. Here we show significant relationships between the telomere lengths of red blood cells and several somatic tissues at adulthood. As red blood cells are easily accessible and suitable for the longitudinal monitoring of the individual rate of telomere loss, our study confirms that telomere length measured in red blood cells could serve as a surrogate for telomere length in the whole avian organism.     

Telomere length measurement-caveats and a critical assessment of the available technologies and tools

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.     

Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement

BACKGROUND: Telomeres are highly conserved repeats at the ends of chromosomes that maintain chromosome stability and reflect the replicative potential of cells. Telomere length can be determined by Southern blot hybridization or quantitative fluorescence in situ hybridization (Q-FISH). Recently, two flow cytometry-based (Flow) FISH protocols have been published. METHODS: We compared the telomere length measured by Southern blotting and Flow FISH using standard beads to calibrate and quantify the fluorescence intensity. RESULTS: The telomeric fluorescence of cord blood and peripheral blood mononuclear cells was similar to that reported by other studies. There was a linear relationship between the telomeric fluorescence determined by Flow FISH and the telomere fragment size determined by Southern blotting (r = 0.89; P < 0.001). CONCLUSION: It is important to set up a center-specific curve and select appropriate cell lines for reference. This Q-Flow FISH protocol will facilitate the measurement of telomere length and allow more meaningful comparison of data (in standard fluorescence units or fragment size) between institutes.     

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.     

Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension

In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.     

Telomere length, chromatin structure and chromosome fusigenic potential

Telomeres are specialized structures at chromosome ends that are thought to function as buffers against chromosome fusion. Several studies suggest that telomere shortening may render chromosomes fusigenic. We used a novel quantitative fluorescence in situ hybridization procedure to estimate telomere length in individual mammalian chromosomes, and G-banding and chromosome painting techniques to determine chromosome fusigenic potential. All analysed Chinese hamster and mouse cell lines exhibited shorter telomeres at short chromosome arms than at long chromosome arms. However, no clear link between short telomeres and chromosome fusigenic potential was observed, i.e. frequencies of telomeric associations were higher in cell lines exhibiting longer telomeres. We speculate that chromosome fusigenic potential in mammalian cell lines may be determined not only by telomere length but also by the status of telomere chromatin structure. This is supported by the observed presence of chromatin filaments linking telomeres in Chinese hamster chromosomes and of multibranched chromosomes oriented end-to-end in the murine severe combined immunodeficient (SCID) cell line. Multibranched chromosomes are the hallmark of the human ICF (Immune deficiency, Centromeric instability, Facial abnormalities) syndrome, characterized by alterations in heterochromatin structure. Received: 13 June 1997; in revised form: 3 August 1997 / Accepted: 4 August 1997     

Epigenetic differences between sister chromatids?

Semi-conservative replication ensures that the DNA sequence of sister chromatids is identical except for replication errors and variation in the length of telomere repeats resulting from replicative losses and variable end processing. What happens with the various epigenetic marks during DNA replication is less clear. Many chromatin marks are likely to be copied onto both sister chromatids in conjunction with DNA replication, whereas others could be distributed randomly between sister chromatids. Epigenetic differences between sister chromatids could also emerge in a more predictable manner, for example, following processes that are associated with lagging strand DNA replication. The resulting epigenetic differences between sister chromatids could result in different gene expression patterns in daughter cells. This possibility has been difficult to test because techniques to distinguish between parental sister chromatids require analysis of single cells and are not obvious. Here, we briefly review the topic of sister chromatid epigenetics and discuss how the identification of sister chromatids in cells could change the way we think about asymmetric cell divisions and stochastic variation in gene expression between cells in general and paired daughter cells in particular.     

Pds5 is required for homologue pairing and inhibits synapsis of sister chromatids during yeast meiosis

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.     

The metaphase scaffold is helically folded: sister chromatids have predominantly opposite helical handedness

We have studied the three-dimensional folding of the scaffolding in histone H1-depleted chromosomes by immunofluorescence with an antibody specific for topoisomerase II. Two different types of decondensed chromosomes are observed. The majority of the chromosomes are expanded, and the central fluorescence signal is surrounded by a large halo of chromatin. A much smaller number of chromosomes are more compact in length; they contain a smaller halo of chromatin and their scaffolds are not extended but folded into a genuine, quite regular helical coil. This conclusion is based on a three-dimensional structural analysis by optical sectioning. The number of helical coils is related to chromosome length. Surprisingly, sister chromatids have predominantly opposite helical handedness; that is, they are related by mirror symmetry.