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The pseudoautosomal region (PAR) is a segment of shared homology between the sex chromosomes. Here we report additional probes for this region of the mouse genome. Genetic and fluorescence in situ hybridization analyses indicate that one probe, PAR-4, hybridizes to the pseudoautosomal telomere and a minor locus at the telomere of chromosome 9 and that a PCR assay based on the PAR-4 sequence amplifies only the pseudoautosomal locus (DXYHgu1). The region detected by PAR-4 is structurally unstable; it shows polymorphism both between mouse strains and between animals of the same inbred strain, which implies an unusually high mutation rate. Variation occurs in the region adjacent to a (TTAGGG)n array. Two pseudoautosomal probes can also hybridize to the distal telomeres of chromosomes 9 and 13, and all three telomeres contain DXYMov15. The similarity between these telomeres may reflect ancestral telomere-telomere exchange.

Pericentric heterochromatin and telomeres have been shown to be capable of repressing the expression of genes located in close proximity. The effect of adjacent structural sequences on gene expression will be important in the design of mammalian artificial chromosomes. In the process of using telomere-containing constructs to generate a deletion panel of the long arm of the human X chromosome, several cell lines were produced which appeared by in situ hybridization to be broken in Xq at or near the centromere. After analysis of end clones rescued from these cell lines, only two produced data consistent with breaks in the alpha satellite array without accompanying rearrangements. The mitotic stability of an X chromosome, with at least 750 kb of the alpha satellite array deleted, was compared to controls where the alpha satellite array remained intact. No significant change in the stability of the chromosome was observed, suggesting that the truncated chromosome has a fully functional mitotic centromere. There was no detectable change in the expression of the hygromycin resistance gene, which is located between a functional centromere and telomere, in this cell line. This study indicates that structural elements flanking a mammalian selectable marker do not result in silencing.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.