Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair

Replicative Age Induces Mitotic Recombination in the Ribosomal RNA Gene Cluster of Saccharomyces cerevisiae

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells

The Elongator Complex Interacts with PCNA and Modulates Transcriptional Silencing and Sensitivity to DNA Damage Agents

Histone chaperones CAF-1 and Asf1 function to deposit newly synthesized histones onto replicating DNA to promote nucleosome formation in a proliferating cell nuclear antigen (PCNA) dependent process. The DNA replication- or DNA repair-coupled nucleosome assembly pathways are important for maintenance of transcriptional gene silencing and genome stability. However, how these pathways are regulated is not well understood. Here we report an interaction between the Elongator histone acetyltransferase and the proliferating cell nuclear antigen. Cells lacking Elp3 (K-acetyltransferase Kat9), the catalytic subunit of the six-subunit Elongator complex, partially lose silencing of reporter genes at the chromosome VIIL telomere and at the HMR locus, and are sensitive to the DNA replication inhibitor hydroxyurea (HU) and the damaging agent methyl methanesulfonate (MMS). Like deletion of the ELP3, mutation of each of the four other subunits of the Elongator complex as well as mutations in Elp3 that compromise the formation of the Elongator complex also result in loss of silencing and increased HU sensitivity. Moreover, Elp3 is required for S-phase progression in the presence of HU. Epistasis analysis indicates that the elp3Δ mutant, which itself is sensitive to MMS, exacerbates the MMS sensitivity of cells lacking histone chaperones Asf1, CAF-1 and the H3 lysine 56 acetyltransferase Rtt109. The elp3Δ mutant has allele specific genetic interactions with mutations in POL30 that encodes PCNA and PCNA binds to the Elongator complex both in vivo and in vitro. Together, these results uncover a novel role for the intact Elongator complex in transcriptional silencing and maintenance of genome stability, and it does so in a pathway linked to the DNA replication and DNA repair protein PCNA

The life and scientific work of William R. Evitt (1923-2009)

Occasionally (and fortunately), circumstances and timing combine to allow an individual, almost singlehandedly, to generate a paradigm shift in his or her chosen field of inquiry. William R. (‘Bill’) Evitt (1923-2009) was such a person. During his career as a palaeontologist, Bill Evitt made lasting and profound contributions to the study of both dinoflagellates and trilobites. He had a distinguished, long and varied career, researching first trilobites and techniques in palaeontology before moving on to marine palynomorphs. Bill is undoubtedly best known for his work on dinoflagellates, especially their resting cysts. He worked at three major US universities and spent a highly significant period in the oil industry. Bill's early profound interest in the natural sciences was actively encouraged both by his parents and at school. His alma mater was Johns Hopkins University where, commencing in 1940, he studied chemistry and geology as an undergraduate. He quickly developed a strong vocation in the earth sciences, and became fascinated by the fossiliferous Lower Palaeozoic strata of the northwestern United States. Bill commenced a PhD project on silicified Middle Ordovician trilobites from Virginia in 1943. His doctoral research was interrupted by military service during World War II; Bill served as an aerial photograph interpreter in China in 1944 and 1945, and received the Bronze Star for his excellent work. Upon demobilisation from the US Army Air Force, he resumed work on his PhD and was given significant teaching duties at Johns Hopkins, which he thoroughly enjoyed. He accepted his first professional position, as an instructor in sedimentary geology, at the University of Rochester in late 1948. Here Bill supervised his first two graduate students, and shared a great cameraderie with a highly motivated student body which largely comprised World War II veterans. At Rochester, Bill continued his trilobite research, and was the editor of the Journal of Paleontology between 1953 and 1956. Seeking a new challenge, he joined the Carter Oil Company in Tulsa, Oklahoma, during 1956. This brought about an irrevocable realignment of his research interests from trilobites to marine palynology. He undertook basic research on aquatic palynomorphs in a very well-resourced laboratory under the direction of one of his most influential mentors, William S. ‘Bill’ Hoffmeister. Bill Evitt visited the influential European palynologists Georges Deflandre and Alfred Eisenack during late 1959 and, while in Tulsa, first developed several groundbreaking hypotheses. He soon realised that the distinctive morphology of certain fossil dinoflagellates, notably the archaeopyle, meant that they represent the resting cyst stage of the life cycle. The archaeopyle clearly allows the excystment of the cell contents, and comprises one or more plate areas. Bill also concluded that spine-bearing palynomorphs, then called hystrichospheres, could be divided into two groups. The largely Palaeozoic spine-bearing palynomorphs are of uncertain biological affinity, and these were termed acritarchs. Moreover, he determined that unequivocal dinoflagellate cysts are all Mesozoic or younger, and that the fossil record of dinoflagellates is highly selective. Bill was always an academic at heart and he joined Stanford University in 1962, where he remained until retiring in 1988. Bill enjoyed getting back into teaching after his six years in industry. During his 26-year tenure at Stanford, Bill continued to revolutionise our understanding of dinoflagellate cysts. He produced many highly influential papers and two major textbooks. The highlights include defining the acritarchs and comprehensively documenting the archaeopyle, together with highly detailed work on the morphology of Nannoceratopsis and Palaeoperidinium pyrophorum using the scanning electron microscope. Bill supervised 11 graduate students while at Stanford University. He organised the Penrose Conference on Modern and Fossil Dinoflagellates in 1978, which was so successful that similar meetings have been held about every four years since that inaugural symposium. Bill also taught many short courses on dinoflagellate cysts aimed at the professional community. Unlike many eminent geologists, Bill actually retired from actively working in the earth sciences. His full retirement was in 1988; after this he worked on only a small number of dinoflagellate cyst projects, including an extensive paper on the genus Palaeoperidinium

Alterations of leptin in the course of inflammation and severe sepsis

<p>Abstract</p> <p>Background</p> <p>The adipokine leptin regulates energy expenditure, vascular function, bone and cartilage growth as well as the immune system and systemic inflammatory response. Several activating effects towards T cells, monocytes, endothelium cells and cytokine production have been reported suggesting a protective role of leptin in the setting of an acute systemic inflammation. However, the pathophysiological role of leptin during severe sepsis is currently not elucidated in detail. This study aims to investigate leptin expression in cultured human adipocytes within an inflammatory model and in patients suffering from severe sepsis and evaluates treatment effects of drotrecogin alpha (activated) (DAA), the recombinant form of human activated protein C.</p> <p>Methods</p> <p>In an <it>in-vitro</it> inflammatory model of adipocyte cell-culture the effect of DAA on leptin mRNA expression was evaluated. Synthesis of mRNA was measured by quantitative polymerase chain reaction (qPCR). Additionally, supernatants of these adipocytes as well as serum levels of adiponectin were measured in blood of 104 severe septic patients by ELISA-method. 26 patients were treated with DAA (DAA+), 78 patients were not treated with DAA (DAA-).</p> <p>Results</p> <p>Stimulation of human adipocytes with TNF alpha over 6 and 24 hours resulted in a significant decrease by 46% and 59% of leptin mRNA transcripts compared to un-stimulated controls (p < 0.05). Leptin levels of supernatants of adipocyte culture decreased by 25% and 23% (p < 0.05) after incubation with TNF alpha after 6 and 24 hours. Incubation with DAA at 50 ng/ml DAA and 5 μg/ml doubled mRNA expression significantly at 24 hours (p < 0.05) but not at 6 hours. From day 1 to day 3 of sepsis, leptin levels increased in DAA+ compared to DAA- patients (p<0.10).</p> <p>Conclusions</p> <p>Leptin appears to be involved in the pathogenesis of a systemic inflammatory response during sepsis. Administration of DAA significantly increased leptin expression. The specific mechanism or even benefit of DAA towards leptin needs further ongoing research.</p

Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length

Almost 400 genes affect yeast telomere length, including Est1, which is critical for recruitment and activation of telomerase. Here we use mass spectrometry to identify novel telomerase regulators by their co-purification with the telomerase holoenzyme. In addition to all known subunits, over 100 proteins are telomerase associated, including all three subunits of the essential Cdc48-Npl4-Ufd1 complex as well as three E3 ubiquitin ligases. The Cdc48 complex is evolutionarily conserved and targets ubiquitinated proteins for degradation. Est1 levels are ∼40-fold higher in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Deletion of the telomerase-associated E3 ligase, Ufd4, in cdc48-3 cells further increases Est1 abundance but suppresses the telomere length phenotype of the single mutant. These data argue that, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1