Chromosome XII context is important for rDNA function in yeast

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of ∼150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6–12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae

MOESM1 of Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator

Additional file 1. Addiitonal figures and tables

Analysis of split-chromosomes in the mutants by CHEF gel electrophoresis and Southern hybridization (A–C)

<p><b>Copyright information:</b></p><p>Taken from "Chromosome XII context is important for rDNA function in yeast"</p><p>Nucleic Acids Research 2006;34(10):2914-2924.</p><p>Published online 31 May 2006</p><p>PMCID:PMC1474064.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Chromosomal DNA was isolated from the following strains: () ID-11, () ID-12 and () ID-13. () Analysis of rDNA copy numbers. Genomic DNA was digested at a unique KpnI site in the rDNA unit and subjected to electrophoresis followed by Southern hybridization using 5S rDNA as a probe. A single copy gene was used as an internal control for normalization. Because the normalization factor for the 5S rDNA probe was found to be 15 relative to the single copy gene, rDNA copy numbers were determined by multiplying by 15. () Relative quantification of 18S rRNA using real-time RT–PCR. The 18S rRNA amount is divided by the ACT1 amount to obtain a normalized 18S rRNA value and the normalized amount of 18S rRNA in FY833 (WT) was used to compare the relative amount of 18S rRNA in different strains