Telomere-associated Sequences Research Articles

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.

Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C base pairs.

We have isolated telomeric DNA and telomere-associated sequences from Chlamydomonas reinhardtii. The terminal telomere sequences of the green alga Chlamydomonas are composed of (TTTTAGGG)n repeats that are similar, but not identical, to those of the higher plant Arabidopsis thaliana. We demonstrate that these repeats are telomeric by their preferential sensitivity to nuclease Bal-31 digestion, their similarity to A. thaliana telomeres, their orientation relative to the end of the chromosome, and the methods used for their isolation. Five independent telomere clones were isolated, and three of these clones include closely related telomere-associated sequences. One of these telomere-associated sequences hybridizes to a number of genomic fragments sensitive to digestion with the exonuclease Bal-31. Like telomere sequences from other organisms, the C. reinhardtii telomeres display a bias for guanine and thymine nucleotides on the 3'-end strand. However, the sequence of Chlamydomonas telomeres is more A + T-rich than any other known telomere sequence. We propose that the common feature of all known telomere is the frequent occurrence of tracts of three or more adjacent guanine residues.

A truncated human chromosome 16 associated with α thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n

The instability of chromosomes with breaks induced by X-irradiation led to the proposal that the natural ends of chromosomes are capped by a specialized structure, the telomere. Telomeres prevent end-to-end fusions and exonucleolytic degradation, enable the end of the linear DNA molecule to replicate, and function in cell division. Human telomeric DNA comprises approximately 2-20 kilobases (kb) of the tandemly repeated sequence (TTAGGG)n oriented 5'----3' in towards the end of the chromosome, interspersed with variant repeats in the proximal region. Immediately subtelomeric lie families of unrelated repeat motifs (telomere-associated sequences) whose function, if any, is unknown. In lower eukaryotes the formation and maintenance of telomeres may be mediated enzymatically (by telomerase) or by recombination; in man the mechanisms are poorly understood, although telomerase has been identified in HeLa cells. Here we describe an alpha thalassaemia mutation associated with terminal truncation of the short arm of chromosome 16 (within band 16p13-3) to a site 50 kb distal to the alpha globin genes, and show that (TTAGGG)n has been added directly to the site of the break. The mutation is stably inherited, proving that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This mechanism may occur in any genetic disease associated with chromosome truncation.

Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector

The construction of a yeast artificial chromosome containing a human DNA insert is reported. This molecule of about 200 kb behaves as a native yeast chromosome since it has a very high mitotic stability and is present in the yeast transformant clone at a copy number similar to that of the resident chromosomes. Hybridization with the TTAGGG sequence demonstrates that this chromosome contains human telomeric sequences. In situ hybridization of the biotin-labelled artificial chromosome to metaphase human chromosomes shows that the insert occupies a telomeric position on the long arm of chromosome 9. Since the fragment was cloned as an EcoRI insert and not as a telomere, it is situated medially to the telomeric sequences and harbours telomere-associated sequences, that have been shown to contain the TTAGGG sequence. The fragment represents the end of the genetic map of chromosome 9 and thus can be used to characterize the sequence and the structure of the chromosomal region that runs from the end of the chromosome to the first gene.

Distribution of telomere-associated sequences on natural chromosomes in Saccharomyces cerevisiae.

Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae. The pattern of X and Y' distribution was different for each strain. At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.

Distribution of telomere-associated sequences on natural chromosomes in Saccharomyces cerevisiae.

Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae. The pattern of X and Y' distribution was different for each strain. At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.

Distribution of telomere-associated sequences in yeast.

Two middle repetitive DNA sequences called X and Y' are found near the telomeres of many chromosomes in Saccharomyces cerevisiae. Orthogonal field gel electrophoresis (OFAGE) was used to examine the distribution of X and Y' on different yeast chromosomes. Although the distribution of X and Y' varies among different laboratory strains of yeast, most yeast chromosomes in four different strains carry both X and Y'. However, at least one chromosome in each strain lacks the Y' element. This result indicates that Y' is not essential for replication or segregation of at least some yeast chromosomes.

Long tandem arrays of complex repeat units in Chironomus telomeres.

A cloned 340-bp DNA fragment excised by EcoRI from the Chironomus pallividittatus genome has been localized to the telomeres by in situ hybridization as well as to connectives between telomeres. No hybridization was observed in other regions of the chromosomes. Another cloned EcoRI fragment, 525 bp long has also been studied. This represents a partial duplication of the 340-bp sequence. Genomic blot hybridization experiments show that the 340-bp sequence is a representative monomeric unit of tandemly repeated arrays which account for 1.2% of the Chironomus genome, on average 300 kb per telomere. The repeat unit contains two types of subrepeats each present twice per repeat unit. Northern blot hybridization experiments show that the telomere-associated sequences are transcribed into a discrete RNA species approximately 20 kb in size. The evolution of this telomere-associated DNA is discussed.