Summary

The genes encoding ribosomal RNA (rDNA) are the most abundant genes in the eukaryotic genome. They reside in tandem repetitive clusters, in some cases totaling hundreds of copies. Due to their repetitive nature, the rDNA loci form some of the most fragile sites in the chromosome (Kobayashi, 2006) In the case of the budding yeast Saccharomyces cerevisiae, the rDNA occupies about 10 % of the total genome and its stability determines that of the whole genome (Kobayashi, 2011a, b). The unusual structure of the rDNA has an impact on cellular functions such as senescence (Kobayashi, 2008 ).
Experimental findings for RNA polymerase I, Fob1 and Sir2 (Kobayashi et al., 1998; Kobayashi et al., 2004) indicate that cells have developed a specialized mechanism to maintain rDNA stability in addition to the general genome maintenance mechanisms. The rDNA occupies ~60% of chromosome XII and the variation in its copy number changes the length of this chromosome which can be determined by pulsed-field gel electrophoresis and used to monitor rDNA stability( Kobayashi et al., 1998; Kobayashi et al., 2004). For example, in the case of a mutant with unstable rDNA the band for chromosome XII becomes broader than in the case of the wild type. The variation in rDNA copy number is furthermore one of the best indicators for a change in overall genome stability.
The genome of S. cerevisiae is composed of ~6,000 genes of which ~4,900 are not essential for viability and can be deleted (Winzeler et al., 1999; Giaever et al., 2002 ). To identify which of these non-essential genes contribute to rDNA stability we screened a library of 4876 deletion strains, by pulsed-field gel electrophoresis for a variation in rDNA copy number (Saka et al., 2016) as listed in the database presented here. It was found that more than 600 genes play a role in the maintenance of the rDNA locus and it can be expected that some of these genes are also important for preserving the overall integrity of the yeast genome.

Methods

A deletion library of S. cerevisiae BY4741 (a S288c background) in which each strain carries a deletion of a specific gene after its ORF has been replaced by a KanMX marker (Wach et al., 1994; Giaever et al., 2002) was obtained from Open Biosystems (Thermo Scientific) and screened by pulsed-field gel electrophoresis to analyse rDNA length and stability, as defined by the rDNA copy number and the variation therein, respectively.
We cultured mutants in 0.5 ml YPD medium at 30°C, harvested the cells and isolated the DNA by the agarose plug method for pulsed-field gel electrophoresis as described before (Ide and Kobayashi, 2010). Pulsed-field gel electrophoresis was performed with a Chef Mapper (Bio-Rad) as described previously (Kobayashi et al., 1998).The gels were stained with ethidium bromide (EtBr) and blotted for Southern analysis. Chromosome XII was detected with an rDNA-specific probe (Kobayashi et al., 1998).

Results and Discussion

The results can be seen in a searchable table above.
The information has been organized using the following categories:

• “ORF name”;
• “Gene name”;
• “rDNA stability”;
• “rDNA copy number”;
• “Gel (Plate/Row/Column)”;
• “Replicative lifespan”; and
• “Other chromosome abnormalities”, which are explained below.

Mutants with an unusual length or stability of rDNA are expected to have a defect in the copy number regulation system. For an in-depth discussion, see Saka et al., 2016

ORF name, Gene name: All analyzed genes are listed by ORF - and gene name and more information on each gene can be retrieved by direct links from the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/; Cherry, et al., 2012).

rDNA stability: The stability of the rDNA in the mutant strains is determined by the sharpness of the chromosome XII band as observed after pulsed-field gel electrophoresis. The strains are ranked in four groups by variability in chromosome XII migration caused by an unstable rDNA copy number. In some cases DNA could not be isolated because of very poor growth so that rDNA stability was not determined (indicated as “N.D.”).

• Rank 1. Strains of rank 1 have a sharper band for chromosome XII than the wild-type strain, indicating a reduced variability in rDNA copy number.
• Rank 2.Chromosome XII in strains of rank 2 is indistinguishable from that of the wild-type strain, ”W”, suggesting that the gene deleted in a rank 2 strain has no role in maintaining rDNA stability. The wild-type “W” is shown in the left side of each gel.
• Rank 3. In the case of strains of rank 3 (3-A-7) the band for chromosome XII is broader than in the case of the wild-type strain. This means that the rDNA in these strains is less stable than in the wild type.
• Rank 4. No discrete band for chromosome XII can be observed for strains of rank 4. In this class of mutants, the rDNA is very unstable with ongoing changes of rDNA copy number like in a sir2 mutant (47-F-2). These strains have the highest rDNA variability.

rDNA copy number: The rDNA copy numbers for mutant strains with relatively stable rDNA (ranking 1 to 3, see above) have been estimated from the length of chromosome XII　as observed by pulsed-field gel electrophoresis and Southern blotting. Four classes have been defined to rank strains according to their number of rDNA copies.

• Class 1. Mutants have less rDNA than the wild type. The copy number is estimated to be below 80.
• Class 2. The rDNA copy number in mutants of class 2 is between 80 and 200, which is comparable to that of the wild-type strain.
• Class 3. Mutants in class 3 have a higher rDNA copy number than the wild type strain. The copy number is estimated to range from 200 to 450.
• Class 4. The rDNA in strains of class 4 is estimated to reach a very high copy number of over 450.
• Note: For mutants with very unstable rDNA (in rank 4 as described above) a discrete band for chromosome XII could not be detected, which is annotated with “-”. In cases that DNA could not be isolated because of poor growth, the rDNA copy number was not determined (“N.D.”).

Gel (Plate/Row/Column): The “Plate/Row/Column” information points to the lane with DNA isolated from the mutant on the linked gel pictures and Southern hybridization patterns which have been used to judge rDNA stability and copy number.

Replicative Lifespan: When available, information from the SGD concerning the replicative lifespan of the deletion mutant is shown and linked to the relevant publications. In the rDNA theory for lifespan, rDNA stability is one of the most important factors that determine the lifespan of a strain (Kobayashi, 2008; 2011a, b)

Other chromosome abnormalities: When observed, abnormalities observed for other chromosomes are described under this heading.

References

• Cherry, J.M. et al. (2012) Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 40, D700-705.
• Ide, S. and Kobayashi, T. (2010) Analysis of DNA replication in Saccharomyces cerevisiae by two-dimensional and pulsed-field gel electrophoresis. Curr Protoc Cell Biol Chapter 22: Unit 22 14.
• Kobayashi, T. (2006) Strategies to maintain the stability of the ribosomal RNA gene repeats--collaboration of recombination, cohesion, and condensation. Genes Genet Syst 81: 155-161.
• Kobayashi, T. (2008) A new role of the rDNA and nucleolus in the nucleus - rDNA instability maintains genome integrity-. BioEssays 30 , 267-272.
• Kobayashi, T. (2011a) Regulation of ribosomal RNA gene copy number and its role in modulating genome integrity and evolutionary adaptability in yeast. Cell. Mol. Life Sci. 68, 1395-1403.
• Kobayashi, T. (2011b) How does genome instability affect lifespan?: roles of rDNA and telomeres. Genes Cells 16, 617-624.
• Kobayashi T, Heck DJ, Nomura M, Horiuchi T (1998) Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev 12, 3821-30.
• Kobayashi, T., Horiuchi, T., Tongaonkar, P., Vu, L., and Nomura, M. (2004) SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast. Cell 117, 441-453.
• Giaever G et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387-391.
• Saka, K., Takahashi, A., Sasaki, M. and Kobayashi, T. (2016) More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance. Nucleic Acids Res., published online, doi: 10.1093/nar/gkw110.
• Wach, A., Brachat, A., Poehlmann, R., and Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast.10, 1793-1808.
• Winzeler EA et al. (1999) Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285, 901-906.

Contributors:

• Kimiko Saka - Pulsed-field electrophoresis, data analysis
• Itamar Nunes - Web arrangement and database development (current version)
• Kanae Fujimaki - Web arrangement (first version)
• Mariko Sasaki - Maintenance
• Takehiko Kobayashi - Data analysis, general coordination

This work was supported in part by grants-in-aid for Scientific Research (23114002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan to TK.
Thanks to Ms. Tokuko Matsumoto (Lafula, co.)

Contact:

Takehiko Kobayashi tako2015@iam.u-tokyo.ac.jp