Induction of the fragile X on BrdU-substituted chromosomes with direct visualization of sister chromatid exchanges on banded chromosomes.
After incorporation of BrdU for one or more replication cycles, the fragile site at Xq27 [fra(X)] was induced by a late pulse with excess thymidine (dT), resulting in the simultaneous visualization of G bands and differentially stained sister chromatids. The degree of BrdU substitution (uni- vs bifilarly substituted DNA) did not affect the expression of hte fra(X). Without addition of dT, expression was the same in M1, M2, and M3 cells. With the addition of dT, expression was reduced in M1 cells and increased in M2 and M3 cells. One way to explain this fact would be an increased repair of the fragile site in M1 cells by illegitimate G:BrdU pairing under dCTP-deficient conditions. A preferential depletion of M3 cells, and to a lesser extent also M2 cells, could suggest a synergistic toxic effect of BrdU substitution and dCTP depletion. With this technique, sister chromatid exchanges (SCEs) could be directly localized at band level, facilitating a more detailed study of SCEs at the Xq27 fragile site.
Tommerup
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Department of Medical Genetics, John F. Kennedy Institute, Glostrup, Denmark.
D001973:Bromodeoxyuridine / Q000494:pharmacology; D002871:Chromosome Banding; D005600:Fragile X Syndrome / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008214:Lymphocytes / Q000648:ultrastructure; D008297:Male; D012729:Sex Chromosome Aberrations / Q000235:genetics*; D012854:Sister Chromatid Exchange; D014960:X Chromosome
D001973:Bromodeoxyuridine