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.

Figure 1.—

Genetic cross and screen for Y, rDNA deletions. In generation 0, females harboring a heat-shock-inducible I-CreI nuclease are mated to males with a recently isogenized yellow⁺-marked Y chromosome. Males were heat-shocked as larvae and crossed to a common yellow white stock en masse in generation 1. Collecting individual males in generation 2 allowed us to sample independent I-CreI-induced rDNA events of the Y chromosomes. Males were crossed to both fresh yellow white females to establish a stock and to C(1)DX females, whose compound-X chromosome lacks rDNA, to determine if damage to the rDNA had occurred. Damage could be assessed as an altered female-to-male ratio (at an extreme, 0:1) or as bobbed female phenotypes (in generation 3). In every subsequent generation, males were backcrossed to the maternal genotype (yellow white) to maintain the stock and C(1)DX females to retest the rDNA array. Genetic nomenclature: hs-I-CreI is P{v^+t1.8=hs-I-CreI.R}2A, v¹; y w is y¹ w^67c23; Y10 is y⁺Yw⁺, Dp(1;Y) y⁺, P{w⁺=RSw}10A, or y⁺Yw⁻, Dp(1;Y) y⁺, P{w⁻=RSw⁻}10B; and C(1)DX is C(1)DX, y¹ f¹ bb⁰.

Figure 2.—

Real-time qPCR to measure rDNA content. (a) Traces of qPCR reactions to amplify the 18S rRNA and tRNA^K genes at three different concentrations of DNA extracted from C(1)DX/Y10B females. Traces show triplicate reactions set in parallel; each triplet is labeled with a letter (a–f), which corresponds to the data in the accompanying graphs. g and h are no-template controls. Bar graphs show the average ± standard error of the mean ranges for amplification cycles (Ct). The rightmost graph shows the difference between rDNA and tDNA threshold cycles (ΔCt) with ranges equal to root pooled squares of standard errors. (b) Traces of qPCR reactions to amplify 18S and tRNA^K genes from wild type (Y10B, traces b and e) and a rDNA deficiency chromosome (l-473, traces i and j). b, e, g, and h are the same traces as in a. Accompanying graphs show Ct for these reactions: the rightmost graph shows ΔCt as a measure of the rDNA/tDNA copy number, and ΔΔCt shows the difference between l-473 and Y10B in Ct, corrected (by the tDNA measurement) for DNA concentration. The difference in rDNA copy number is 2^ΔΔCt. The y-axes are either cycles of qPCR or differences in cycles between different samples.

Figure 3.—

Y, rDNA^Df allelic series. qPCR was used to measure rDNA array size in the alleles generated in this work. (a) 10B is the progenitor chromosome. The remainder are the recovered alleles, sorted by average size. Data are presented as the average ± standard error of the mean. Chromosome names indicate their phenotype (wt, wild-type; bb, bobbed; l, lethal) as the sole source of rDNA in the organism, as well as their allele number. The approximate range that corresponds to those phenotypes is indicated at the left of the graph. The y-axis is ratio of wild-type Y10B chromosome rDNA content. (b) Bobbed flies were photographed and are presented in order of decreasing rDNA array size (taken from a), which correlates with an increasing severity of the bobbed phenotype.

Figure 4.—

rDNA arrays undergo slow gradual magnification as well as sporadic fast magnification. Six chromosomes were monitored every generation by selecting four to eight individuals for rDNA array size measurement. The data for each individual are shown (x's), as well as the average of the population (connected by lines). Solid data points indicate individuals with the same phenotype (lethal or bobbed) as previous generations, and shaded data points are from individuals whose phenotype changed (to wild type for bb-465 or to bobbed for l-473). The x-axes are the successive generations after establishment as stock. The y-axes are the ratios to wild-type Y10B chromosome rDNA content. For clarity, standard errors are not depicted.

Figure 5.—

rDNA arrays undergo magnification when exposed to I-CreI. (a) Y chromosomes with previously reduced rDNA arrays were exposed to I-CreI induced by heat shock. This cross is similar to the one described in Figure 1, but here we screened for reversion of the lethal-bobbed phenotype to the bobbed or wild-type phenotype. A control cross was performed in parallel with X chromosomes without the I-CreI-expressing transgene. (b) Results of rDNA quantification. Each graph contains data showing the relative average for Y10B (defined as 1.00) and the parental chromosome prior to heat-shock induction of I-CreI, both as solid data points. Shaded data points are confidence intervals for individuals (the average of replicate qPCR reactions with standard errors of the mean), and photographs of a subset of those individuals show the bobbed phenotype. The final data point, l-481-rev*, is from the control cross, which did not express I-CreI. The large amount of rDNA is most consistent with nondisjunction producing a C(1)DX/Y10B, rDNA^Df/Y, rDNA⁺ individual.