Defects in the NuA4 acetyltransferase complex increase stability of the ribosomal RNA gene and extend replicative lifespan.

Genome instability is a cause of cellular senescence. The ribosomal RNA gene repeat (rDNA) is one of the most unstable regions in the genome and its instability is proposed to be a major inducer of cellular senescence and restricted lifespan. We previously conducted a genome-wide screen using a budding yeast deletion library to identify mutants that exhibit a change in the stability of the rDNA region, compared to the wild-type. To investigate the correlation between rDNA stability and lifespan, we examined deletion mutants with very stable rDNA and found that deletion of EAF3, encoding a component of the NuA4 histone acetyltransferase complex, reproducibly resulted in increased stabilization of the rDNA. In the absence of Eaf3, and of other subunits of the NuA4 complex, we observed lower levels of extrachromosomal rDNA circles that are produced by recombination in the rDNA and are thus an indicator of rDNA instability. The replicative lifespan in the eaf3 mutant was extended by ~30%, compared to the wild-type strain. Our findings provide evidence that rDNA stability is correlated with extended replicative lifespan. The eaf3 mutation possibly affects the non-coding transcription in rDNA that regulates rDNA recombination through cohesin dissociation.

Existence, Transition, and Propagation of Intermediate Silencing States in Ribosomal DNA.

The MET3 promoter (MET3pr) inserted into the silenced chromosome in budding yeast can overcome Sir2-dependent silencing upon induction and activate transcription in every single cell among a population. Despite the fact that MET3pr is turned on in all the cells, its activity still shows very high cell-to-cell variability. To understand the nature of such "gene expression noise," we followed the dynamics of the MET3pr-GFP expression inserted into ribosomal DNA (rDNA) using time-lapse microscopy. We found that the noisy "on" state is comprised of multiple substable states with discrete expression levels. These intermediate states stochastically transition between each other, with "up" transitions among different activated states occurring exclusively near the mitotic exit and "down" transitions occurring throughout the rest of the cell cycle. Such cell cycle dependence likely reflects the dynamic activity of the rDNA-specific RENT complex, as MET3pr-GFP expression in a telomeric locus does not have the same cell cycle dependence. The MET3pr-GFP expression in rDNA is highly correlated in mother and daughter cells after cell division, indicating that the silenced state in the mother cell is inherited in daughter cells. These states are disrupted by a brief repression and reset upon a second activation. Potential mechanisms behind these observations are further discussed.

Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles.

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India

Abstract Yellow pigmented, filamentous, Gram-negative bacteria belonging to genus Flavobacterium are commonly associated with infections in stressed fish. In this study, inter-species diversity of Flavobacterium was studied in apparently healthy freshwater farmed fishes. For this, ninety one yellow pigmented bacteria were isolated from skin and gill samples (n = 38) of three farmed fish species i.e. Labeo rohita, Catla catla and Cyprinus carpio. Among them, only twelve bacterial isolates (13.18%) were identified as Flavobacterium spp. on the basis of morphological, biochemical tests, partial 16S rDNA gene sequencing and phylogenetic analysis. On the basis of 16S rDNA gene sequencing, all the 12 isolates were 97.6-100% similar to six different formally described species of genus Flavobacterium. The 16S rDNA based phylogenetic analysis grouped these strains into six different clades. Of the 12 isolates, six strains (Fl9S1-6) grouped with F. suncheonense, two strains (Fl6I2, Fl6I3) with F. indicum and the rest four strains (Fl1A1, Fl2G1, Fl3H1 and Fl10T1) clustered with F. aquaticum, F. granuli, F. hercynium and F. terrae, respectively. None of these species except, F. hercynium were previously reported from fish. All the isolated Flavobacterium species possessed the ability of adhesion and biofilm formation to colonize the external surface of healthy fish. The present study is the first record of tropical freshwater farmed fishes as hosts to five environmentally associated species of the Flavobacterium.

Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions.

In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.

Cellular senescence in yeast is regulated by rDNA noncoding transcription.

Genomic instability is a conserved factor in lifespan reduction, although the molecular mechanism is not known. Studies in the yeast Saccharomyces cerevisiae over the past 20 years have found a connection between the ribosomal RNA gene cluster (rDNA) and lifespan. The highly repetitive rDNA exhibits genomic instability, and the antiaging histone deacetylase gene SIR2 regulates this instability. We previously proposed that SIR2 governs lifespan by repressing rDNA noncoding transcription and rDNA instability, but the extent to which lifespan is affected by SIR2 acting at the rDNA versus other genomic regions, and the relationship between rDNA noncoding transcription/rDNA stability and lifespan have remained controversial. To control rDNA noncoding transcription and rDNA instability, we use a strain in which the rDNA noncoding promoter is replaced with an inducible promoter. Here, we show that repression of noncoding transcription extends lifespan and makes SIR2 dispensable for lifespan extension. These results indicate that Sir2 maintains lifespan through repression of E-pro noncoding transcription in the rDNA cluster, rather than pleiotropically at other loci. The observation of rDNA instability in other organisms, including humans, suggests that this may be a conserved aging pathway.

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

Principles of chromosomal organization: lessons from yeast.

The spatial organization of genes and chromosomes plays an important role in the regulation of several DNA processes. However, the principles and forces underlying this nonrandom organization are mostly unknown. Despite its small dimension, and thanks to new imaging and biochemical techniques, studies of the budding yeast nucleus have led to significant insights into chromosome arrangement and dynamics. The dynamic organization of the yeast genome during interphase argues for both the physical properties of the chromatin fiber and specific molecular interactions as drivers of nuclear order.

[Genotypic analysis and plant growth-promoting ability of four plant growth-promoting bacteria from mangrove].

OBJECTIVE: We identified four strains of plant growth-promoting bacteria (PGPB) and their plant growth-promoting ability. METHODS: Four PGPB strains were genetically analyzed by PCR detection of nifH and 16S rRNA gene. Phosphate-solubilizing and nitrogen-fixation capacity were examined by spectrophotometric quantification and acetylene reduction assay, respectively. Effect of strain inoculation on plant growth was also evaluated. RESULTS: Phylogenetic analysis based on nifH and 16S rRNA gene sequences indicated that strain HN011 was mostly related to Vibrio natriegens, and SZ7-1 and SZ7-2 resembled Klebsiella oxytoca. Although similarity of 16S rRNA sequence showed that SZ002 belongs to Paenibacillus sp., nifH gene of SZ002 had high sequence similarity with Klebsiella genus. Phosphate solubilization showed that insoluble phosphate was well solubilized in the liquid medium by all four strains of PGPB, which also had high nitrogen-fixation capacity. Plant dry weight, total N and total P were higher in some inoculated than in the non-inoculated plants (P < 0.05). CONCLUSION: Our results showed that all four strains of PGPB isolated from mangrove had both phosphate solubilization and nitrogen fixation ability, resulting in beneficial effects on growth.

Candida albicans, a distinctive fungal model for cellular aging study.

The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.

Epifluorescence and light microscopy evidencing structural and functional polymorphism of ribosomal DNA in fish (Teleostei: Astyanax fasciatus).

New karyotypic data are presented for the Astyanax fasciatus species complex from four different locations on the Upper Tibagi River in the state of Paraná, Brazil. Chromosome markers were analyzed using conventional (Ag-NOR) and molecular (FISH with 18S biotinylated probes) methods. Two cytotypes were found in cell counts with diploid number 2n=48 chromosomes and 2n=50 chromosomes, previously denominated Cytotype A and B, respectively. Two specific patterns of Ag-NORs markers (ribosomal gene activity) were found, with intra-population and inter-population variations. Cytotype A exhibited two to three chromosomes with NOR sites in the metaphases analyzed. In Cytotype B specimens, up to three markers were found, demonstrating greater intra-population and inter-population variation. All individuals with only one chromosome pair with NORs were located in the telomeric region of the short arm of Chromosome 5. This characteristic was interpreted as ancestral for the species. Another identified pattern revealed a site in the telomeric region probably in the long arm of Chromosome 4 and another submetacentric chromosome with bitelomeric marks exclusively in specimens with 2n=50 chromosomes. In the FISH analysis (ribosomal gene structure), five to seven markers were identified in Cytotype A and three to seven markers were identified in Cytotype B. Structural chromosome events and/or transposable elements are required to explain the ribosomal gene location diversity in these organisms. The results of the present study corroborate the hypothesis that the A. fasciatus of the Upper Tibagi River region constitute a species complex.

Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis.

The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

Nuclear myosin I acts in concert with polymeric actin to drive RNA polymerase I transcription.

Actin is associated with all three nuclear RNA polymerases and acts in concert with nuclear myosin I (NM1) to drive transcription. Practically nothing is known regarding the state of actin and the functional interplay of actin and NM1 in transcription. Here we show that actin and NM1 act in concert to promote RNA polymerase I (Pol I) transcription. Drugs that prevent actin polymerization or inhibit myosin function inhibit Pol I transcription in vivo and in vitro. Mutants that stabilize the polymeric state actin are tightly associated with Pol I and activate transcription, whereas a polymerization-deficient mutant does not bind to Pol I and does not promote rDNA transcription. Consistent with nuclear actin and myosin synergizing in transcription activation, NM1 mutants that lack specific functions, such as binding to ATP, actin, or calmodulin, are incapable of associating with Pol I and rDNA. The results show that actin polymerization and the motor function of NM1 are required for association with the Pol I transcription machinery and transcription activation. These observations provide insights into the cooperative action of actin and myosin in the nucleus and reveal an actomyosin-based mechanism in transcription.

The nucleolus: a model for the organization of nuclear functions.

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.

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 approximately 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.

Comparison of Dam tagging and chromatin immunoprecipitation as tools for the identification of the binding sites for S. pombe CENP-C.

We have established the identity of the Schizosaccharomyces pombe homologue of vertebrate CENP-C and Saccharomyces cerevisiae MIF2p and have used it to compare Dam tagging and chromatin immunoprecipitation (ChiP)as tools for the mapping of protein binding sites on DNA. ChiP shows that S. pombe CENP-C binds to the central core and inner repeats of the S. pombe centromere. It binds weakly, however, to the outer repeats. The binding pattern is thus similar to that of S. pombe CENP-A. Dam-tagged S. pombe CENP-C, however, methylates the entire centromere and 5 kb of flanking DNA. This comparison suggests that Dam tagging is less precise as a tool for mapping DNA binding sites than ChiP. We have also used the Dam tagging technique to address the question of whether there is any CENP-C binding to the ribosomal DNA in S. pombe and find none.

SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions.

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.