Summary In eukaryotic genomes, ribosomal DNA (rDNA) generally resides as a highly repetitive and dynamic structure, making it difficult to study. Here, a synthetic rDNA array on chromosome III in budding yeast was constructed to serve as the sole source of rRNA. Utilizing the loxPsym site within each rDNA repeat and the Cre recombinase, we were able to reduce the copy number to as few as eight copies. Additionally, we constructed strains with two or three rDNA arrays, and found that the presence of multiple arrays did not affect the formation of a single nucleolus. Although alteration on the position and number of rDNA arrays did impact three-dimensional genome structure, the additional rDNA arrays had no deleterious influence on cell growth or transcriptomes. Together, this study sheds light on the high plasticity of rDNA organization and opens up opportunities for future rDNA engineering. Highlights A method was established for efficient construction of synthetic rDNA arrays in budding yeast The rDNA repeats in a haploid yeast can be reduced to as few as eight copies to support cell viability Yeast cells with two or three DNA arrays on distinct chromosomes form a single nucleolus. Dispersed rDNA arrays result in no deleterious influence on cell growth or transcriptomes.

High plasticity of ribosomal DNA organization in budding yeast