Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

Journal article

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.

Authors: Warren, S; Knight, S; Peters, J; Stayton, C; Consalez, G

• Journal: Proceedings of the National Academy of Sciences of the United States of America
• Volume: 87
• Issue: 10
• Pages: 3856-3860
• Publication date: 1990-05-01
• DOI: 10.1073/pnas.87.10.3856
• EISSN: 1091-6490
• ISSN: 0027-8424
• Language: English
• Keywords: Cell Survival; Nucleic Acid Hybridization; Sex Chromosome Aberrations; Clone Cells; Cell Line; X Chromosome; Hypoxanthine Phosphoribosyltransferase; Humans; Chromosome Banding; DNA; Animals; Hybrid Cells; Glucosephosphate Dehydrogenase; DNA Probes; Blotting, Southern; Fragile X Syndrome