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.     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.     

Conditioned medium from amniotic cells protects striatal degeneration and ameliorates motor deficits in the R6/2 mouse model of Huntington's disease

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro‐inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM‐hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune‐based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM‐hAMSC display less severe signs of neurological dysfunction than saline‐treated ones. CM‐hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM‐hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM‐hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM‐hAMSC could act by modulating inflammatory cells, and more specifically microglia.     

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Background

Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.

Methodology/Principal Findings

In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.

Conclusions/Significance

This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.     

Characterization of taxonomically restricted genes in a phylum-restricted cell type

Background  

Despite decades of research, the molecular mechanisms responsible for the evolution of morphological diversity remain poorly understood. While current models assume that species-specific morphologies are governed by differential use of conserved genetic regulatory circuits, it is debated whether non-conserved taxonomically restricted genes are also involved in making taxonomically relevant structures. The genomic resources available in Hydra, a member of the early branching animal phylum Cnidaria, provide a unique opportunity to study the molecular evolution of morphological novelties such as the nematocyte, a cell type characteristic of, and unique to, Cnidaria.     

Long telomeric C-rich 5'-tails in human replicating cells

Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3'-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3'-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5'-tail, the incompletely replicated template of the leading strand. We examined the presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3'-overhang, the 5'-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5' C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening.     

DNA-DNA hybridization evidence of the rapid rate of muroid rodent DNA evolution

Single-copy nuclear DNAs (scnDNAs) of eight species of arvicoline and six species of murine rodents were compared using DNA-DNA hybridization. The branching pattern derived from the DNA comparisons is congruent with the fossil evidence and supported by comparative biochemical, chromosomal, and morphological studies. The recently improved fossil record for these lineages provides seven approximate divergence dates, which were used to calibrate the DNA-hybridization data. The average rate of scnDNA divergence was estimated as 2.5%/Myr. This is approximately 10 times the rate in the hominoid primates. These results agree with previous reports of accelerated DNA evolution in muroid rodents and extend the DNA-DNA hybridization data set of Brownell.      

Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas

We have sequenced the small-subunit ribosomal RNA gene of the amoebo- flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.      

Evolutionary rates for tuf genes in endosymbionts of aphids

The gene encoding elongation factor Tu (tuf) in aphid endosymbionts (genus Buchnera) evolves at rates of 1.3 x 10(-10) to 2.5 x 10(-10) nonsynonymous substitutions and 3.9 x 10(-9) to 8.0 x 10(-9) synonymous substitutions per position per year. These rates, which are at present among the most reliable substitution rates for protein-coding genes of bacteria, have been obtained by calibrating the nodes in the phylogenetic tree produced from the Buchnera EF-Tu sequences using divergence times for the corresponding ancestral aphid hosts. We also present data suggesting that the rates of nonsynonymous substitutions are significantly higher in the endosymbiont lineages than in the closely related free-living bacteria Escherichia coli and Salmonella typhimurium. Synonymous substitution rates for Buchnera approximate estimated mutation rates for E. coli and S. typhimurium, as expected if synonymous changes act as neutral mutations in Buchnera. We relate the observed differences in substitution frequencies to the absence of selective codon preferences in Buchnera and to the influence of Muller's ratchet on small asexual populations.