A study on nucleolar DNA: isolation of DNA from fibrillar components and ultrastructural localization of different DNA probes.

The nature and localization of DNA contained in the fibrillar centres and the dense fibrillar component (the fibrillar complex) in the nucleoli, was studied in human LEP cells, Sertoli cells, spermatogonia A and in mitotic chromosomes of stimulated lymphocytes. A novel procedure for isolating the intact fibrillar complex from LEP cells was used; the complex contains DNA that hybridizes to secondary constrictions of mitotic chromosomes and to 28 S rDNA sequences, on Southern blots. Electron microscopic DNA-DNA in situ hybridization was performed, with (a) a probe prepared from DNA extracted from the fibrillar complex of LEP cells, (b) a probe for human total genomic DNA, and (c) a probe for the transcribed part of human rDNA. On the basis of the results obtained we conclude that the ribosomal RNA genes in human Sertoli cells and spermatogonia A are predominantly associated with the dense fibrillar component, including the border region between fibrillar centres and the dense fibrillar component. The ribosomal RNA genes are the main, if not exclusive, DNA type present in the fibrillar complex in the studied cell types.