Objectif
By electroporating telomeric constructs into a hybrid line carrying a human X chromosome and applying selections it was possible to isolate a number of cell lines which were candidates for lines which carried terminally deleted X chromosomes. These were analysed further. It was established that these cell lines, represented unrearranged terminal deletions of the X chromosome and terminal restriction mapping and the process of cloning terminal restriction fragments was started.
A number of clones were isolated and sequence data derived from them. The clones or the sequence tagged site (STS) primer pairs derived from their sequences were mapped back on the panel of hybrid lines. The terminal genomic sequences from 80% of the end clones mapped more proximally than the deletion point determined by marker loss in the cell line from which they were derived. It was revealed that the original genomic restriction fragment was retained in addition to the fragment derived from it by addition of a telomere. Taken together these results imply an inverted terminal duplication of the chromosome.
In the majority of the duplicated lines the duplications were found to be between 2 megabase pairs (Mbp) and 5 Mbp in size. The duplications make these cell lines unsuitable for deriving terminal restriction maps. There was no effect on the positioning of centromeric and telomeric parts of a chromosome when moving a telomere from its normal position on the chromosome to a previously internal position. No measurable effect was found on the fidelity of chromosome segregation when the long arm of the X chromosome was deleted.
STSs were mapped on the breakpoint panel. Mapping on the panel demonstrated that the end clones were not telomeric: they were all contained in telomere hybrids with more proximal breakpoints.
The STSs were used to identify yeast artificial chromosome (YAC) clones from the total human YAC library. YACs were also isolated using STS derived from known markers which were mapped on the telomere hybrid panel and used to map breakpoints in Xq.
The biological properties of telomeres will be applied to mapping human chromosomes. Telomeres are the ends of eukaryotic chromosomes. Introduction of telomeres by recombination at interstitial locations on yeast chromosomes leads to breakage at these sites at a rate of about 10{-2} per cell per generation. Reintroduction of telomeric constructs into mammalian cells has the same result. After breakage only the chromosome fragment which retains the centromere is maintained in the cell.
This provides a powerful means of creating deleted chromosomes which are tagged at one end by the introduced telomere. These chromosomes will be used to produce a physical and genetic map of the human X chromosome. Telomeric constructs will be introduced into a hybrid cell line containing a human X chromosome. These constructs will contain a telomere and a marker gene selectable in the mammalian cell. In the case of the X chromosome the distal location of the HPRT gene means that we can select cells which were originally HPRT plus, and which have now become HPRT minus. A hybrid cell line has been made in which the X chromosome can be maintained in the absence of selection for HPRT. These cells will be cloned, characterized and used as a set of large scale nested deletions of the X chromosome.
Molecular methods will be used to confirm, for each clone, that the loss of the HPRT gene has occurred by breakage at an introduced telomere. By in situ hybridization a location for the breakpoint will be established and will be refined using available STS markers for the X chromosome long arm. Unambiguous partial digest maps can be created at these telomeres (using the selectable marker as a probe) since the map extends in only one direction.
The distance which can be mapped from a single site is currently about 5 Mbp. The X chromosome long arm is about 100 Mbp in length and so could, in principle, be mapped physically using 20 clones with evenly spaced X chromosome breakpoints. Complete coverage will require the analysis of many more cell lines but the practicality of the approach would be adequately demonstrated by the analysis of about 20 lines. Probes derived from regions of the X chromosome immediately proximal to the introduced telomere and selectable marker can be derived either by rescue of DNA fragments in E. coli or by inverse PCR.
These probes will be used to isolate YAC clones from existing libraries. These YACs will be screened for the presence of microsatellite hypervariable repeats and flanking PCR primers established. The genetic map around these markers will be determined enabling a close correlation between it and the genetic map to be established rapidly. This is a powerful new approach which is generally applicable to mapping human chromosomes and which complements other more established techniques.
Champ scientifique (EuroSciVoc)
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CORDIS classe les projets avec EuroSciVoc, une taxonomie multilingue des domaines scientifiques, grâce à un processus semi-automatique basé sur des techniques TLN. Voir: Le vocabulaire scientifique européen.
- sciences naturelles sciences biologiques génétique ADN
- sciences naturelles sciences biologiques génétique chromosome
- sciences naturelles sciences biologiques génétique génome génome eucaryote
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