Non-thalamic origin of zebrafish sensory nuclei implies convergent evolution of visual pathways in amniotes and teleosts.

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homology is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a thalamic-like sensory structure of teleosts, the preglomerular complex (PG), focusing on the visual projection neurons. Similarly to the tectofugal thalamic nuclei in amniotes, the lateral nucleus of PG receives tectal information and projects to the pallium. However, our cell lineage study in zebrafish reveals that the majority of PG cells are derived from the midbrain, unlike the amniote thalamus. We also demonstrate that the PG projection neurons develop gradually until late juvenile stages. Our data suggest that teleost PG, as a whole, is not homologous to the amniote thalamus. Thus, the thalamocortical-like projections evolved from a non-forebrain cell population, which indicates a surprising degree of variation in the vertebrate sensory systems.

More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance.

Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal.

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.

Rtt109 prevents hyper-amplification of ribosomal RNA genes through histone modification in budding yeast.

The genes encoding ribosomal RNA are the most abundant in the eukaryotic genome. They reside in tandem repetitive clusters, in some cases totaling hundreds of copies. Due to their repetitive structure, ribosomal RNA genes (rDNA) are easily lost by recombination events within the repeated cluster. We previously identified a unique gene amplification system driven by unequal sister-chromatid recombination during DNA replication. The system compensates for such copy number losses, thus maintaining proper copy number. Here, through a genome-wide screen for genes regulating rDNA copy number, we found that the rtt109 mutant exhibited a hyper-amplification phenotype (∼3 times greater than the wild-type level). RTT109 encodes an acetyl transferase that acetylates lysine 56 of histone H3 and which functions in replication-coupled nucleosome assembly. Relative to unequal sister-chromatid recombination-based amplification (∼1 copy/cell division), the rate of the hyper-amplification in the rtt109 mutant was extremely high (>100 copies/cell division). Cohesin dissociation that promotes unequal sister-chromatid recombination was not observed in this mutant. During hyper-amplification, production level of extra-chromosomal rDNA circles (ERC) by intra-chromosomal recombination in the rDNA was reduced. Interestingly, during amplification, a plasmid containing an rDNA unit integrated into the rDNA as a tandem array. These results support the idea that tandem DNA arrays are produced and incorporated through rolling-circle-type replication. We propose that, in the rtt109 mutant, rDNA hyper-amplification is caused by uncontrolled rolling-circle-type replication.

The effect of replication initiation on gene amplification in the rDNA and its relationship to aging.

In eukaryotes, the ribosomal DNA (rDNA) consists of long tandem repeat arrays. These repeated genes are unstable because homologous recombination between them results in copy number loss. To maintain high copy numbers, yeast has an amplification system that works through a pathway involving the replication fork barrier site and unequal sister chromatid recombination. In this study, we show that an active replication origin is essential for amplification, and the amplification rate correlates with origin activity. Moreover, origin activity affects the levels of extrachromosomal rDNA circles (ERC) that are thought to promote aging. Surprisingly, we found that reduction in ERC level results in shorter life span. We instead show that life span correlates with rDNA stability, which is preferentially reduced in mother cells, and that episomes can induce rDNA instability. These data support a model in which rDNA instability itself is a cause of aging in yeast.

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.