Expression of I-CreI Endonuclease Generates Deletions Within the rDNA of Drosophila

Abstract

The rDNA arrays in Drosophila contain the cis-acting nucleolus organizer regions responsible for forming the nucleolus and the genes for the 28S, 18S, and 5.8S/2S RNA components of the ribosomes and so serve a central role in protein synthesis. Mutations or alterations that affect the nucleolus organizer region have pleiotropic effects on genome regulation and development and may play a role in genomewide phenomena such as aging and cancer. We demonstrate a method to create an allelic series of graded deletions in the Drosophila Y-linked rDNA of otherwise isogenic chromosomes, quantify the size of the deletions using real-time PCR, and monitor magnification of the rDNA arrays as their functions are restored. We use this series to define the thresholds of Y-linked rDNA required for sufficient protein translation, as well as establish the rate of Y-linked rDNA magnification in Drosophila. Finally, we show that I-CreI expression can revert rDNA deletion phenotypes, suggesting that double-strand breaks are sufficient to induce rDNA magnification.

Ribosomal DNA contributes to global chromatin regulation

“…Based on the ''bobbed'' phenotype, which manifests as a result of limited translational capacity in protein-synthesis-intensive tissues (such as cuticular and bristle secreting cells), we divided deletions into two categories: ''small deletions'' limited for rRNA production and expressing a bobbed phenotype, and ''large deletions'' incapable of providing sufficient rRNA when the sole source of rDNA in the cell and expressing a bobbed-lethal phenotype. These categories were confirmed using real-time PCR to measure the number of rDNA cistrons in the array (34).…”

“…YrDNA deletion strains are described in Paredes and Maggert (34), but for ease have been given different names here, which Dissection. Larvae were raised on standard cornmeal molasses fly food supplemented with baker's yeast and raised at 18°C.…”

“…DNA Preparations. DNA was extracted from single larval or adult flies as described in Paredes and Maggert (34). DNA was quantified using a Nanodrop and diluted to 10 ng/ L. Triplicate real-time PCR reactions were performed with 10 ng template.…”

“…One microliter from the sonicated sample was used in each of triplicate real-time PCR reactions. Primers, controls, and data analyses are described in Paredes and Maggert (34).…”

“…The deletions we generated possess the ability to magnify at a rate of up to 15 copies per fly generation (34), which provided us the opportunity to confirm the correlation between expression and rDNA deletion. We observed expression in six strains as they magnified and simultaneously measured the quantity of rDNA.…”