Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Physical Activity and Sedentary Behavior in Relation to Esophageal and Gastric Cancers in the NIH-AARP Cohort

Introduction

Body mass index is known to be positively associated with an increased risk of adenocarcinomas of the esophagus, yet there is there limited evidence on whether physical activity or sedentary behavior affects risk of histology- and site-specific upper gastrointestinal cancers. We used the NIH-AARP Diet and Health Study to assess these exposures in relation to esophageal adenocarcinoma (EA), esophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma (GCA), and gastric non-cardia adenocarcinoma (GNCA).

Methods

Self-administered questionnaires were used to elicit physical activity and sedentary behavior exposures at various age periods. Cohort members were followed via linkage to the US Postal Service National Change of Address database, the Social Security Administration Death Master File, and the National Death Index. Cox proportional hazards regression models were used to estimate hazard ratios (HR) and 95 percent confidence intervals (95%CI)

Results

During 4.8 million person years, there were a total of 215 incident ESCCs, 631 EAs, 453 GCAs, and 501 GNCAs for analysis. Strenuous physical activity in the last 12 months (HR_{>5 times/week vs. never}=0.58, 95%CI: 0.39, 0.88) and typical physical activity and sports during ages 15–18 years (p for trend=0.01) were each inversely associated with GNCA risk. Increased sedentary behavior was inversely associated with EA (HR_{5–6 hrs/day vs. <1 hr}=0.57, 95%CI: 0.36, 0.92). There was no evidence that BMI was a confounder or effect modifier of any relationship. After adjustment for multiple testing, none of these results were deemed to be statistically significant at p<0.05.

Conclusions

We find evidence for an inverse association between physical activity and GNCA risk. Associations between body mass index and adenocarcinomas of the esophagus do not appear to be related to physical activity and sedentary behavior.     

SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

Centromeric histone H3, CENP-A^Cse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A^Cse4 restricts its localization to centromeres. Mislocalization of CENP-A^Cse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.     

Thermo-osmoregulation of Heat-Labile Enterotoxin Expression by <Emphasis Type="Italic">Escherichia coli</Emphasis>

Abstract:Enterotoxigenic Escherichia coli causes diarrhea by producing several virulence factors including heat-labile enterotoxin (LT). LT is maximally expressed at 37°C. The histone-like nucleoid structuring protein (H-NS) appears to inhibit LT expression by binding to a downstream regulatory element (DRE) at low temperatures. An hns⁺ E. coli strain, X7026, carrying an LT–beta-galactosidase translational fusion plasmid (pLT-lac) was shown to be responsive to varying amounts of sodium chloride (NaCl) as well as sucrose or lithium chloride. Maximal responsiveness to the various osmolytes was obtained with cells grown at 37°C under microaerophilic conditions. Temperature-osmotic upshift experiments demonstrate LT expression is thermo-osmoregulated. pLT-lac was tested in an hns strain or its congenic hns⁺ strain for its response to NaCl. LT expression is elevated in the hns strain regardless of NaCl concentration and retains its osmoresponsiveness. The response of the DRE deletion plasmid (pLT-lacNC) to NaCl is similar to that of the undeleted plasmid.     

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.     

Recognizing chromosomes in trouble: association of the spindle checkpoint protein Bub3p with altered kinetochores and a unique defective centromere

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.     

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. 222Ra), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of 3He2+ ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups (> or = two-fold over controls) and was comparable whether cells were traversed by one or two 3He2+ ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer.