Sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in human cells: The role of transcription units

Abstract

DNA sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in HeLa cells have been cloned and 106 representative clones sequenced; 10 clones containing random genomic fragments sewed as controls. As chromatin is prone to rearrangement, care was taken to isolate sequences using 'physiological' conditions that did not create additional attachments. Comparison (by Southern blotting) of the concentration of each cloned sequence in 'total' and 'attached' fractions of DNA showed that most clones did contain attached sequences, but even highly-attached sequences were not attached in all cells in the population. Results demonstrated that 28% of clones were derived from three specific parts of the mitochondrial genome and 22% from different parts of the alu repeat. In addition, 41% of clones contained unique nuclear sequences; these contained no more of the motifs found attached to nuclear scaffolds or matrices (ie SARs or MARs) than would be expected from their base composition. No other attachment motif(s) could be identified by sequence analysis. However, Northern blotting showed that all the mitochondrial clones and 76% of clones containing unique sequences were transcribed; the degree of attachment correlated with transcriptional activity. These results are consistent with transcription being responsible for ever-changing attachments in both nuclei and mitochondria.