Preparative dual‐beam sorting of the human Y chromosome and in situ hybridization of cloned DNA probes

Bivariate Hoechst/chromomycin flow karyotypes for chromosomes from a Chinese hamster‐human hybrid cell line (CH‐Y‐VII) were established that have retained a human Y chromosome. These bivariate flow karyotypes showed the human Y chromo‐some to be completely separated from the peaks for the Chinese hamster chromosomes. In preparative dual‐beam sorting: experiments, 3 × 10⁶ chromosomes were sorted from the Y peak into frozen petri dishes. An examination of Q‐banded samples of sorted chromosomes revealed that 82% ± 5% of them were human Y chromosomes. The DNA from the sorted chromosomes (approximately 250 ng) was isolated and used to establish a genomic library (vector λ gt WES.λB). Three clones (YACG 45, 52, 54) of this library containing inserts of repetitive human DNA were used for chromosomal localization by means of in situ hybridization to metaphase spreads of male human lymphocytes and of CH‐Y‐VII cells. In all three cases, a significant binding to the human Y chromosome was observed. A more detailed study of the chromosomal distribution of sequences homologous to the insert of YACG 45 suggested the existence of minor binding sites on several human autosomes. Southern blot analysis revealed the existence of other human specific sequences without Y chromosome specificity.