Identification and sorting of micronuclei containing individual chromosomes.

Micronuclei are diploid genome subfractions which can be induced by colchicine treatment. The purpose of the present study was to identify the chromosome content of micronuclei. Accordingly, PtK1 cells were micronucleated with colchicine and their DNA was labeled by the Hoechst 33342 fluorochrome. The fluorescence intensity of the micronuclei was measured by flow cytometry and compared to that of metaphase chromosomes of the same species. We observed histograms exhibiting five distinct peaks for both micronuclei and chromosomes. Each class of micronuclei corresponded to a peak in the flow karyotype with the same values. As the smallest micronuclei contained the same amount of DNA as one chromosome, it seemed possible to identify the DNA content of the micronuclei containing a single chromosome. This identification was confirmed by fluorescence microscope observations of the micronuclei and chromosomes sorted from their respective peaks. This was clear for peak number 3, which exhibited fluorescent markers both in micronuclei and in chromosomes. These markers, called chromocenters, were connected with the nucleoli in micronuclei and were specific for the nucleolar organizer regions of the X chromosomes. As we paid particular attention to maintaining the transcriptional activity of the micronuclei throughout the procedure used to sort and identify them, it is concluded that such an approach might be very suitable for investigating the expression of reduced nuclear domains corresponding to individual chromosomes.