Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina Japonica

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing. The total length of 45S rDNA repeat unit of S. japonica was 8995 bp, including 5420 bp of 18s-5.8s-25s rDNA and 3575 bp of IGS (Intergenic Spacer), with the GC content of 51.4%. IGS was composed of 465 bp 3’-outer transcribed spacer (ETS), 874 bp 5’-ETS, and 2236 bp non transcribed spacer (NTS), with the GC content of 50.1%.Fiber-FISH (fiber-fluorescence in situ hybridization, fiber-FISH) analysis of 45S rDNA on the BAC molecule of female gametophytes of S. japonica illustrated that each fiber had at least five continuous moniliform hybridization signal points, indicating the distribution of 45S rDNA repeat unit on the bacterial artificial chromosome. This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S. japonica, and the successful fiber-FISH analysis of 45S rDNA on BAC molecule would contribute to the construction of the physical map and Map-based cloning of this kelp.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

Age-Dependent Ribosomal DNA Variations in Mice

ABSTRACT The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast Saccharomyces cerevisiae is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.

Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

The ribosomal RNA gene, which consists of tandem repetitive arrays (rDNA repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in ageing mammalian cells. Using quantitative analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations among the repeats with two mutations being unique to old mice. These sequences were conserved in budding yeast and equivalent mutations shortened the yeast chronological lifespan. Our findings suggest that rDNA is also fragile in mammalian cells and alterations within this region have a profound effect on cellular function.Author SummaryThe ribosomal RNA gene (rDNA) is one of the most unstable regions in the genome due to its tandem repetitive structure. rDNA copy number in the budding yeast increases and becomes unstable as the cell ages. It is speculated that the rDNA produces an “aging signal” inducing senescence and death. However, it is unclear how the rDNA repeat changes during the aging process in mammalian cells. In this study, we attempted to identify the age-dependent alteration of rDNA in mice. Using quantitative single cell analysis, we show that rDNA copy number increases in old mice bone marrow cells. By contrast, the level of ribosomal RNA production was reduced because of increased levels of DNA methylation that represses transcription. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice and two of them were found in the conserved region in budding yeast. We then established a yeast strain with the old mouse-specific mutations and found this shortened the lifespan of the cells. These findings suggest that rDNA is also fragile in mammalian cells and alteration to this region of the genome affects cellular senescence.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

The adjustment of transcription and translation rates to variable needs is of utmost importance for the fitness and survival of living cells. We have previously shown that the global transcription rate for RNA polymerase II is regulated differently in cells presenting symmetrical or asymmetrical cell division. The budding yeast Saccharomyces cerevisiae adopts a particular strategy to avoid that the smaller daughter cells increase their total mRNA concentration with every generation. The global mRNA synthesis rate lowers with a growing cell volume, but global mRNA stability increases. In this paper, we address what the solution is to the same theoretical problem for the RNA polymerase I synthesis rate. We find that the RNA polymerase I synthesis rate strictly depends on the copy number of its 35S rRNA gene. For cells with larger cell sizes, such as a mutant cln3 strain, the rDNA repeat copy number is increased by a mechanism based on a feed-back mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of the rRNA genes at the rDNA locus in a volume-dependent manner.

Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.Author SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the “ribosomal DNA locus”, a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.

Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata

We investigated intra- and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2–3 different mtDNA haplotypes. In contrast, Pax-C and newly identified single-copy nuclear genes showed either no sequence differences or minor variations in SNP frequencies segregating among the colonies. Our data suggest past mitochondrial introgression in M. capitata, whereas nuclear single-copy loci show limited variation, highlighting the divergent evolutionary histories of these coral DNA markers.

Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata

We investigated intra and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2-3 different mtDNA haplotypes. In contrast, Pax-C and newly identified single-copy nuclear genes showed either no sequence differences or minor variations in SNP frequencies segregating among the colonies. Our data suggest past mitochondrial introgression in M. capitata, whereas nuclear single-copy loci show limited variation, highlighting the divergent evolutionary histories of these coral DNA markers.

Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata

We investigated intra and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2-3 different mtDNA haplotypes. In contrast, Pax-C and newly identified single-copy nuclear genes showed either no sequence differences or minor variations in SNP frequencies segregating among the colonies. Our data suggest past mitochondrial introgression in M. capitata, whereas nuclear single-copy loci show limited variation, highlighting the divergent evolutionary histories of these coral DNA markers.