Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing

Pericentrlc heterochromatin and telomeres have been shown tobe capable of repressing the expression of genes located Inclose proximity. The effect of adjacent structural sequenceson gene expression will be important in the design of mammalianartificial chromosomes. In the process of using telomere-containlngconstructs to generate a deletion panel of the long arm of thehuman X chromosome, several cell lines were produced which appearedby 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 satellitearray without accompanying rearrangements. The mitotic stabilityof an X chromosome, with at least 750 kb of the alpha satellitearray deleted, was compared to controls where the alpha satellitearray remained Intact. No significant change In the stabilityof the chromosome was observed, suggesting that the truncatedchromosome has a fully functional mitotic centromere. Therewas no detectable change in the expression of the hygromycinresistance gene, which is located between a functional centromereand telomere, In this cell line. This study indicates that structuralelements flanking a mammalian selectable marker do not resultin silencing.