Molecular differentiation of sex chromosomes probed by comparative genomichybridization

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simul taneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-l DNA or unlabelled DNA of the homogam etic sex. CGH visualized the molecular differentiation of the X and Y in th e house mouse, Mus musculus, and in Drosophila melanogaster: while autosome s were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respective ly. There was no differential staining of the X and Y chromosomes in the fl y Megaselia scalaris, indicating an early stage of sex chromosome different iation in this species. In the human and the house mouse, labelled DNA of m ales in the presence of unlabelled DNA of females was sufficient to highlig ht Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, t he silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax mo th Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-deri ved probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuo us. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The con spicuous binding of the male-derived probe to the W chromosomes is presumab ly due to an accumulation of common interspersed repetitive sequences.