Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.

Abstract

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG),, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.Specialized telomeric DNA sequences are required for the replication and stability of linear chromosomes (for review, see ref. 1). In many organisms, telomeric DNA is also associated with recombinational events that result in frequent rearrangements of telomere-proximal DNA (for examples, see refs. 2-4). Two classes of repeated DNA elements have been implicated in these functional attributes of telomeres. The first is a simple repeat sequence with a consensus motif containing a G-rich strand (1); this sequence motif has been found at the termini of linear chromosomes of representative plants, animals, protists, and fungi (1,5,6). Particularly striking is the recent demonstration that the terminal-repeat sequence in the human, (TJAGGG)n, is identical to that in trypanosomes and similar to that in yeast, (TG1_3)", and Tetrahymena, (TTGGGG)n (6).The terminal-repeat sequence is apparently maintained in Tetrahymena macronuclei and in Oxytricha by the templateindependent addition of repeat nucleotides through the enzymatic activity of a ribonucleoprotein termed telomerase (7-9). The Tetrahymena telomerase can recognize oligonucleotides corresponding to the G-rich strand of DNA from terminal-repeat sequences of many organisms, suggesting similar mechanisms of telomere maintenance among these organisms and a strong evolutionary conservation of this process (7,8). Recombination may also be involved in the maintenance of the terminal-repeat sequences in Saccharomyces cerevisiae (10).The second class of repeated telomeric DNA element is localized just proximal to the terminal repeat and is speciesspecific. These subtelomeric repeats are not required for telomere replication and maintenance but are involved in the enhanced recombination often associated with telomeres. In S. cerevisiae, two subtelomeric repeat elements have been characterized, X and Y'. X is present at most ofthe telomeres in a single copy. Y' is present on about half of the telomeres, is sometimes present in multiple copies on a single telomere, and is always distal to X (refs. 11 (15)(16)(17), but their proximity to the chromosomal termini has not been determined at the molecular level. Stu...

A Physical Map of Human Chromosome 7: An Integrated YAC Contig Map with Average STS Spacing of 79 kb

“…); 7q telomere YAC: HTY146 (yWSS4191), GDB: 171469 (Riethman et al 1989)]. Note that the first three components listed above correspond to the ''human chromosome 7 YAC resource'' described previously , whereas the last two components represent subsequent additions.…”

“…The latter was assessed by the systematic examination of CEPH-Genethon mapping data (Cohen et al 1993;. c Miscellaneous YACs mostly isolated from the total human genomic Washington University YAC library (Burke et al 1987;Brownstein et al 1989), as well as a small number of clones derived from the total human genomic ICI YAC library (Anand et al 1990) and a human telomere-specific YAC library (Riethman et al 1989 . Table 1 were summed and the relative totals assessed.…”

Characterization of Physical Gap Sizes at Human Telomeres

“…PCR screening was carried out using STSs developed from either distal markers, telomeric sequence information, half-YAC vector-insert junction sequences, or end sequences from telomeric clones. The methods for identifying half-YAC clones, isolating vector-insert junction fragments, and obtaining sequence information have been described previously (Cross et al 1989;Riethman et al 1989;Negorev et al 1994). Because the subtelomeric repeats are known to be shared among nonhomologous chromosomes, each primer set used for PCR screening was first tested and optimized on a monochromosomal hybrid panel to identify conditions that would produce chromosome-specific PCR amplification for screening purposes.…”

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

“…Interestingly, the human telomere sequence (TAACCC)7 also formed complexes with the plant (GGGTTTA)6, yeast (TGTGTGGG)5, and Oxytricha (GGGGTTTT)5 telomere sequences, as well as the related repetitive sequences (GGTA)10 and (GGGTA)8, although the duplexes were 10-28°C less stable and exhibited lower hyperchromicities than the human-Paramecium telomere complexes ( Table 2). The ability of these divergent telomere sequences to form stable hydrogen-bonded complexes under physiologically relevant conditions (Table 2) may explain their functional interchangeability as telomeres in yeast artificial chromosomes (23,24), and as primers for telomerase activity (4).…”