Vertebrate Telomere Research Articles

Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides.

DNA minor groove-binding compounds (polyamides) that target insect and vertebrate telomeric repeats with high specificity were synthesized. Base pair recognition of these polyamides is based on the presence of the heterocyclic amino acids pyrrole and imidazole. One compound (TH52B) interacts uniquely and with excellent specificity (K(d) = 0.12 nM) with two consecutive insect-type telomeric repeats (TTAGG). A related compound, TH59, displays high specificity (K(d) = 0.5 nM) for tandem vertebrate (TTAGGG) and insect telomeric repeats. The high affinity and specificity of these compounds were achieved by bidentate binding of two flexibly linked DNA-binding moieties. Epifluorescence microscopy studies show that fluorescent derivatives of TH52B and TH59 stain insect or vertebrate telomeres of chromosomes and nuclei sharply. Importantly, the telomere-specific polyamide signals of HeLa chromosomes co-localize with the immunofluorescence signals of the telomere-binding protein TRF1. Our results demonstrate that telomere-specific compounds allow rapid estimation of relative telomere length. The insect-specific compound TH52 was shown to be incorporated rapidly into growing Sf9 cells, underlining the potential of these compounds for telomere biology and possibly human medicine.

Cloning of Molluscan Telomere DNA with (TTAGGG)n Repeat and its Chromosomal Location in the Freshwater Snail Biwamelania habei

Abstract In the present study, we have cloned and determined the nucleotide sequence of telomeric DNA of a freshwater gastropod Biwamelania habei. The sequence was comprised of (TTAGGG)n tandem repeat, which is identical with the sequence of vertebrate telomere. The characteristic sequences, together with the chromosomal localization revealed by FISH analysis and the Bal 31 exonuclease sensitivity of the hybridization signal, support the sequences to be located at chromosomal ends. This is the first report for identification of molluscan telomere DNA and is also the first report of invertebrate telomere with (TTAGGG)n sequence. Although the telomere sequence of B. habei is different from that of any other invertebrates reported to date, the sequence universally occurs among various eukaryotes including some metazoans. It is thus evident that B. habei retains ancestral sequence for invertebrate telomeres. Interstitial telomeric signals were observed in some chromosomes. Some of such interstitial telomeric ...

Insertion of a telomere repeat sequence into a mammalian gene causes chromosome instability.

Telomere repeat sequences cap the ends of eucaryotic chromosomes and help stabilize them. At interstitial sites, however, they may destabilize chromosomes, as suggested by cytogenetic studies in mammalian cells that correlate interstitial telomere sequence with sites of spontaneous and radiation-induced chromosome rearrangements. In no instance is the length, purity, or orientation of the telomere repeats at these potentially destabilizing interstitial sites known. To determine the effects of a defined interstitial telomere sequence on chromosome instability, as well as other aspects of DNA metabolism, we deposited 800 bp of the functional vertebrate telomere repeat, TTAGGG, in two orientations in the second intron of the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. In one orientation, the deposited telomere sequence did not interfere with expression of the APRT gene, whereas in the other it reduced mRNA levels slightly. The telomere sequence did not induce chromosome truncation and the seeding of a new telomere at a frequency above the limits of detection. Similarly, the telomere sequence did not alter the rate or distribution of homologous recombination events. The interstitial telomere repeat sequence in both orientations, however, dramatically increased gene rearrangements some 30-fold. Analysis of individual rearrangements confirmed the involvement of the telomere sequence. These studies define the telomere repeat sequence as a destabilizing element in the interior of chromosomes in mammalian cells.

The solution structure and internal motions of a fragment of the cytidine-rich strand of the human telomere

We present the solution structure of d(CCCTA2CCCTA2CCCTA2CCCT), a fragment of the vertebrate telomere which folds intramolecularly. The four cytidine stretches form an i-motif which includes six intercalated C·C+ pairs and terminates with the cytidines at the 5′ extremity of each stretch. Above, the second TA2 linker loops across one of the narrow grooves, while at the bottom, the first and third linkers loop across the wide grooves.At 30 °C, the spectra of the first and third linkers are quasi-degenerate. Severe broadening at lower temperature indicates that this results from motional averaging between at least two structures of each bottom loop, and makes it impossible to solve the configuration of the bottom loops directly, in contrast to the rest of the structure.We therefore turned to the modified sequence d(CCCTA25mCCCTA2CCCUA2CCCT) in which the two base substitutions (underlined) break the quasi-symmetry between linkers 1 and 3. The three loops follow approximately the hairpin “second pattern” of Hilbers. In the first loop, T4 is in the syn orientation, whereas its analog in the third loop, U16, oriented anti, is in a central location, where it interacts with bases of both loops, thus contributing to their tight association. The only motion is a syn anti flip of A18 in the third loop.Returning to the telomere fragment, we show that each of the bottom loops switches between the structures identified in the first and third loops of the modified structure. The motions are concerted, and the resulting configurations of the bottom loop cluster present a bulge to either right (T4 syn) or left (T16 syn).

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

Intramolecular i-Motif Structures of Telomeric DNA

The i-motif is an intercalated structure formed by association in a head to tail orientation of two parallel duplexes whose strands are held together by hemiprotonated C·C+ pairs. The i-motif may be formed by a single strand containing four cytidine repeats, by association of two strands containing two cytidine repeats or by four strands containing a single cytidine stretch. The repeated C-rich sequences of centromeric and telomeric regions can potentially fold into an intramolecular i-motif. We have investigated by NMR spectroscopy the structure of d(CCCTA2CCCTA2CCCTA2CCCT), a fragment of the vertebrate telomere. It includes an i-motif core of six intercalated C·C+ pairs. At one end (the “top”), the central TA2 linker loops across one of the narrow grooves, and the core is extended by base stacking in the loop. At the bottom, where the two other TA2 linkers loop across the wide grooves, the NMR spectra reveal motions in the microsecond to millisecond scale. The pseudo-symmetry of the structure, which results in degenerate spectra and poor resolution, was broken by appropriate substitution of T by U and of C by 5-methylcytidine (5mC). This allowed us to solve the structure of d(CCCTA25mCCCTA2CCCUA2CCCT). The motion is restricted to a flip of A18 around the glycosidic bond. Returning to the pseudo-symmetrical sequence, we find that each of the bottom loops switches between the structures of the first and third loops of the non-symmetrical sequence. We also analyzed the effects of the loop sequence and of the length of the C-stretches on the topology and stability of the intramolecular i-motif structure.

Telomeres in warm-blooded vertebrates are composed of GC-rich isochores.

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.

Telomeres and telomerase: broad effects on cell growth

During the past year, major advances have been made in understanding the link between telomerase expression and cell immortality. Studies of yeast telomeres have revealed an unexpected role for the non-homologous end-joining machinery in telomere maintenance and have provided the first definitive evidence that telomeres play a critical role in meiosis. Identification of new telomere proteins has led to a better understanding of vertebrate telomere structure and function.

Condensation of Rat Telomere-specific Nucleosomal Arrays Containing Unusually Short DNA Repeats and Histone H1

Vertebrate telomeres contain arrays of nucleosomes with unusually short and regular repeat lengths (Makarov, V. L., Lejnine, S., Bedoyan, J., and Langmore, J. P.(1993) Cell 73, 775-787; Lejnine, S., Makarov, V., and Langmore, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2393-2397). In order to better define the specific structural features of telomere chromatin, we examined the condensation and H1 content of telomere nucleoproteins from rat liver. Velocity sedimentation analysis shows that telomeric nucleosome arrays condense with increasing ionic strength and molecular weight in a manner comparable with that of bulk chromatin despite the very short repeat length. However, these condensed structures do not exhibit the approximately 100-base pair deoxyribonuclease II repeat characteristic of condensed bulk chromatin. Frictional coefficient calculations suggest that telomere-specific higher order structure is more compact than bulk chromatin. Nucleoprotein gel electrophoresis shows that telomeric dinucleosomes from soluble chromatin contain H1. Finally, direct isolation and analysis of telomere nucleoproteins from formaldehyde-cross-linked nuclei indicate the presence of core histone proteins and H1. These results are consistent with the view that a major fraction of the long telomeres of rat are organized as specialized nucleosome arrays with features similar but not identical to those of bulk chromatin.

In search of vertebrate telomeric proteins

Abstract Indirect evidence suggests that vertebrate chromosome ends carry a protective nucleoprotein cap that contains specific telomeric proteins. Telomeric proteins could explain the stringent sequence requirements for de novo telomere formation, aspects of telomere length regulation, the unusual chromatin structure detectable in some vertebrate telomeres, and the attachment of telomeric DNA to the nuclear matrix. Although telomeric proteins have been identified in unicellular organisms, vertebrate telomeric proteins are not well characterized. The search for these factors is discussed in this review.

Telomeric sequences of Asellus aquaticus (Crust. Isop.)

The repeated sequence TTAGGG is present at all tested vertebrate telomeres including those of humans and at the telomeres of evolutionarily very distant organisms such as trypanosomes and slime moulds. We tested for the presence of this sequence in the genome of the crustacean isopod Asellus aquaticus. Fluorescence in situ hybridization and BAL31 nuclease digestion demonstrate that the (TTAGGG)n sequence occurs at the extreme termini of the chromosomes and also at an interstitial site.

A yeast protein that binds to vertebrate telomeres and conserved yeast telomeric junctions.

We have identified three yeast proteins that bind to poly(C.A)/poly(T.G) repeats characteristic of telomeric sequences from yeast to human. TBF alpha binds to the telomeric sequences of yeast, Tetrahymena, and vertebrates. In contrast, TBF beta binds only to yeast telomeric sequences. Also identified was RAP1, the transcriptional silencer protein, which binds to a sequence motif found in upstream activating sequences (UASs) of a number of genes; the sequence motif also occurs frequently in yeast telomeric sequences. Because poly(C.A)/poly(T.G) sequences from a wide range of organisms will serve as the primer for the in vivo extension of telomeres in yeast, TBF alpha is of particular interest. DNase I footprinting analysis indicated that TBF alpha binds to the junction between the subtelomeric X sequence and poly(C1-3A) in a cloned yeast telomere. Examination of the junctions of known X sequences indicated that they all contain one or more repeats of CCCTAA, a sequence that is repeated in vertebrate telomeres. Earlier, Murray et al. (1988) reported that heterologous telomeric sequences positioned as far as several hundred base pairs from the termini of linear molecules can allow the addition of yeast telomeric sequences from nontelomeric termini in vivo. A possible function for TBF alpha might be to serve as an anchoring protein for the yeast telomerase by binding to the conserved junction sequence at a distance from the terminus to allow addition of an irregular repeating sequence at the chromosome end.

Conservation of the human telomere sequence (TTAGGG)n among vertebrates.

To determine the evolutionary origin of the human telomere sequence (TTAGGG)n, biotinylated oligodeoxynucleotides of this sequence were hybridized to metaphase spreads from 91 different species, including representative orders of bony fish, reptiles, amphibians, birds, and mammals. Under stringent hybridization conditions, fluorescent signals were detected at the telomeres of all chromosomes, in all 91 species. The conservation of the (TTAGGG)n sequence and its telomeric location, in species thought to share a common ancestor over 400 million years ago, strongly suggest that this sequence is the functional vertebrate telomere.