Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome

Abstract

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S–28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S–28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a–e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S–28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S–28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.Key words: micronuclei, rDNA, rRNA, single chromosome, in situ hybridization.