Genetic interaction network of the Saccharomyces cerevisiae type 1 phosphatase Glc7

<p>Abstract</p> <p>Background</p> <p>Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous cellular processes in budding yeast.</p> <p>Results</p> <p>We created a novel <it>glc7 </it>catalytic mutant (<it>glc7-E101Q</it>). Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal cell cycle progression and chromosome segregation. This suggests that <it>glc7-E101Q </it>is a hypomorphic <it>glc7 </it>mutant. Synthetic Genetic Array analysis of <it>glc7-E101Q </it>revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with <it>GLC7 </it>was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the interconnection among the modules reflects the cooperation of the processes buffering Glc7 function.</p> <p>Conclusion</p> <p>We found 245 genes required for the normal growth of the <it>glc7-E101Q </it>mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.</p

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples

Community Surveillance of Omicron in Ontario: Wastewater-based Epidemiology Comes of Age

Wastewater-based surveillance of SARS-CoV-2 RNA has been implemented at building, neighbourhood, and city levels throughout the world. Implementation strategies and analysis methods differ, but they all aim to provide rapid and reliable information about community COVID-19 health states. A viable and sustainable SARS-CoV-2 surveillance network must not only provide reliable and timely information about COVID-19 trends, but also provide for scalability as well as accurate detection of known or unknown emerging variants. Emergence of the SARS-CoV-2 variant of concern Omicron in late Fall 2021 presented an excellent opportunity to benchmark individual and aggregated data outputs of the Ontario Wastewater Surveillance Initiative in Canada; this public health-integrated surveillance network monitors wastewaters from over 10 million people across major population centres of the province. We demonstrate that this coordinated approach provides excellent situational awareness, comparing favourably with traditional clinical surveillance measures. Thus, aggregated datasets compiled from multiple wastewater-based surveillance nodes can provide sufficient sensitivity (i.e., early indication of increasing and decreasing incidence of SARS-CoV-2) and specificity (i.e., allele frequency estimation of emerging variants) with which to make informed public health decisions at regional- and state-levels.Ontario Ministry of the Environment, Conservation and Parks|| Genome Canada and Ontario Genomics (OGI-209)||NSERC (ALLRP 555041-20 to C.O.)||Ontario Clean Water Agenc

A role for the «Saccharomyces cerevisiae» kinetochore protein Ame1 in cell cycle control and MT-kinetochore attachment

High fidelity chromosome segregation in all cells requires the formation of bi-oriented attachments between spindle microtubules (MT) and chromosomes. The kinetochore provides a bridge between the MTs and chromosomes. Ame1 is an essential but undercharacterized component of the central kinetochore COMA sub-complex (Ctf19, Okp1, Mcm21, Ame1). In order to characterize Ame1, I used two conditional alleles of the COMA, ame1-4 and okp1-5. I examined the role of Ame1 in the context of the kinetochore and in the maintenance of the spindle assembly checkpoint (SAC) and the formation and repair of kinetochore-MT attachments. I found that ame1-4 cells have a compromised COMA. In contrast, the COMA is disrupted in okp1-5 cells, but Ame1 remains localized to the kinetochore. Nonetheless, the stability of DNA binding and MT binding kinetochore complexes remains intact in ame1-4 and okp1-5 cells. I used the difference between okp1-5 and ame1-4 to further delineate the relationship between Ame1 and the COMA. ame1-4 cells exhibit defective sister chromatid attachments that are not repaired, and are unable to maintain a checkpoint arrest. We find that disruption of the COMA results in a failure to maintain the localization of Sli15 to the kinetochore. In turn, Sli15 functions in checkpoint maintenance and spindle passenger protein migration. Indicative of the loss of passenger protein migration, ame1-4 cells exhibit a cytokinesis defect. Finally, over-expression of OKP1 in ame1-4 cells restores localization of ame1-4p and re-establishes checkpoint maintenance but does not restore Sli15 kinetochore localization. A synthetic genetic screen (SGA) was carried out to identify genetic interactors of the ame1-4 allele. Thirty-three (33) genes were found to interact with ame1-4 to produce a synthetic sick/lethal phenotype. Many genes identified were common interactors with other kinetochore subunits. Comparing the genetic interaction network of the COMA genes, ame1-4 shared many interacDans toute cellule en division, la haute fidélité de la ségrégation des chromosomes passe par la formation d'attachements bi orientés entre les microtubules du fuseau (MT) et les chromosomes. Le kinétochore est la structure faisant le lien entre les MT et les chromosomes. Le complexe COMA (Ctf19, Okp1, Mcm21, Ame1) est un sous-complexe central du kinétochore, et Ame1 en est un composant essentiel quoique peu caractérisé. Dans le but de caractériser Ame1, j'ai utilisé deux allèles du complexe COMA: ame1-4 et okp1-5. J'ai examiné le rôle d'Ame1 dans le cadre du kinétochore et de la maintenance du point de contrôle de l'assemblage du fuseau ainsi que dans la formation et la réparation des attachements entre kinetochore et MT. J'ai constaté que, dans les cellules ame1-4, le complexe COMA est compromis. Dans les cellules okp1-5, le complexe COMA est également perturbé, alors qu'Ame1 est localisé au kinétochore. Toutefois, dans les cellules ame1-4 comme dans les cellules okp1-5, la stabilité de la liaison à l'ADN et aux MT des complexes du kinétochore demeure intacte. J'ai utilisé la différence entre les mutants okp1-5 et ame1-4 pour mieux comprendre la relation entre Ame1 et le complexe COMA. Dans les cellules ame1-4, les attachements des chromatides soeurs sont déficients. Ceux-ci ne sont pas réparés et sont incapables de maintenir l'arrêt de croissance suite à l'activation du point de contrôle. Nous avons constaté que l'inactivation du complexe COMA provient de l'incapacité à localiser Sli15 au kinétochore. Par ailleurs, Sli15 joue un rôle dans la maintenance du point de contrôle et la migration des protéines passagères du fuseau. Le fait que les cellules ame1-4 présentent une cytokinèse déficiente indique une perte de migration des protéines passagères. Enfin, la surexpression d'OKP1 dans les cellules ame1-4 rétablit la localisation de ame1-4 et la maintenance du point de contrôle mais pas la localisation de Sli15 au

The characterization of the approximated locus in D. melanogaster

Bibliography: p. 147-15

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

<p>To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1α associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1α/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.</p

Dynamic regulation of histone H3K9 is linked to the switch between replication and transcription at the Dbf4 origin-promoter locus

<p>The co-regulation of DNA replication and gene transcription is still poorly understood. To gain a better understanding of this important control mechanism, we examined the DNA replication and transcription using the Dbf4 origin-promoter and Dbf4 pseudogene models. We found that origin firing and Dbf4 transcription activity were inversely regulated in a cell cycle-dependent manner. We also found that proteins critical for the regulation of replication (ORC, MCM), transcription (SP1, TFIIB), and cohesin (Smc1, Smc3) and Mediator functions (Med1, Med12) interact with specific sites within and the surrounding regions of the Dbf4 locus in a cell cycle-dependent manner. As expected, replication initiation occurred within a nucleosome-depleted region, and nucleosomes flanked the 2 replication initiation zones. Further, the histone H3 in this region was distinctly acetylated or trimethylated on lysine 9 in a cell cycle-dependent fluctuation pattern: H3K9ac was most prevalent when the Dbf4 transcription level was highest whereas the H3K9me3 level was greatest during and just after replication. The KDM4A histone demethylase, which is responsible for the H3K9me3 modification, was enriched at the Dbf4 origin in a manner coinciding with H3K9me3. Finally, HP1γ, a protein known to interact with H3K9me3 in the heterochromatin was also found enriched at the origin during DNA replication, indicating that H3K9me3 may be required for the regulation of replication at both heterochromatin and euchromatin regions. Taken together, our data show that mammalian cells employ an extremely sophisticated and multilayered co-regulation mechanism for replication and transcription in a highly coordinated manner.</p

Dynamic regulation of histone H3K9 is linked to the switch between replication and transcription at the Dbf4 origin-promoter locus

<p>The co-regulation of DNA replication and gene transcription is still poorly understood. To gain a better understanding of this important control mechanism, we examined the DNA replication and transcription using the Dbf4 origin-promoter and Dbf4 pseudogene models. We found that origin firing and Dbf4 transcription activity were inversely regulated in a cell cycle-dependent manner. We also found that proteins critical for the regulation of replication (ORC, MCM), transcription (SP1, TFIIB), and cohesin (Smc1, Smc3) and Mediator functions (Med1, Med12) interact with specific sites within and the surrounding regions of the Dbf4 locus in a cell cycle-dependent manner. As expected, replication initiation occurred within a nucleosome-depleted region, and nucleosomes flanked the 2 replication initiation zones. Further, the histone H3 in this region was distinctly acetylated or trimethylated on lysine 9 in a cell cycle-dependent fluctuation pattern: H3K9ac was most prevalent when the Dbf4 transcription level was highest whereas the H3K9me3 level was greatest during and just after replication. The KDM4A histone demethylase, which is responsible for the H3K9me3 modification, was enriched at the Dbf4 origin in a manner coinciding with H3K9me3. Finally, HP1γ, a protein known to interact with H3K9me3 in the heterochromatin was also found enriched at the origin during DNA replication, indicating that H3K9me3 may be required for the regulation of replication at both heterochromatin and euchromatin regions. Taken together, our data show that mammalian cells employ an extremely sophisticated and multilayered co-regulation mechanism for replication and transcription in a highly coordinated manner.</p

Cytotoxic and Anti-Plasmodial Activities of Stephania dielsiana Y.C. Wu Extracts and the Isolated Compounds

International audienc