scholarly journals Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Corey Nislow ◽  
Anna Y. Lee ◽  
Patricia L. Allen ◽  
Guri Giaever ◽  
Andrew Smith ◽  
...  
Keyword(s):  
Redox State ◽  
Biological Effects ◽  
Model Organism ◽  
Single Copy ◽  
Complete Collection ◽  
Dna Damaging Agents ◽  
Genome Wide ◽  
Unbiased Manner ◽  
High Concordance ◽  
Yeast Deletion

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Quantitative Trait Loci for the Monoamine-Related Traits Heart Rate and Headless Behavior inDrosophila melanogaster

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 283-294 ◽  
Author(s):  
Kristie Ashton ◽  
Ana Patricia Wagoner ◽  
Roland Carrillo ◽  
Greg Gibson
Keyword(s):  
Heart Rate ◽  
Genetic Variation ◽  
Recombinant Inbred Lines ◽  
Model Organism ◽  
Interval Mapping ◽  
Activity Levels ◽  
Environmental Variance ◽  
Genome Wide ◽  
A Genome ◽  
Monoamine Receptors

AbstractDrosophila melanogaster appears to be well suited as a model organism for quantitative pharmacogenetic analysis. A genome-wide deficiency screen for haploinsufficient effects on prepupal heart rate identified nine regions of the genome that significantly reduce (five deficiencies) or increase (four deficiencies) heart rate across a range of genetic backgrounds. Candidate genes include several neurotransmitter receptor loci, particularly monoamine receptors, consistent with results of prior pharmacological manipulations of heart rate, as well as genes associated with paralytic phenotypes. Significant genetic variation is also shown to exist for a suite of four autonomic behaviors that are exhibited spontaneously upon decapitation, namely, grooming, grasping, righting, and quivering. Overall activity levels are increased by application of particular concentrations of the drugs octopamine and nicotine, but due to high environmental variance both within and among replicate vials, the significance of genetic variation among wild-type lines for response to the drugs is difficult to establish. An interval mapping design was also used to map two or three QTL for each behavioral trait in a set of recombinant inbred lines derived from the laboratory stocks Oregon-R and 2b.

scholarly journals A toolbox of Stable Integration Vectors (SIV) in the fission yeast Schizosaccharomyces pombe

2019 ◽  
Author(s):  
Aleksandar Vještica ◽  
Magdalena Marek ◽  
Pedro N’kosi ◽  
Laura Merlini ◽  
Gaowen Liu ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Model Organism ◽  
Experimental System ◽  
Single Copy ◽  
Cell Physiology ◽  
Large Set ◽  
Stable Integration ◽  
Wide Range ◽  
Fluorescent Tags

AbstractSchizosaccharomyces pombe is a widely used model organism that resembles higher eukaryotes in many aspects of cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

scholarly journals ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

2018 ◽  
Author(s):  
Gabriel Balmus ◽  
Domenic Pilger ◽  
Julia Coates ◽  
Mukerrem Demir ◽  
Matylda Sczaniecka-Clift ◽  
...  
Keyword(s):  
Genetic Resistance ◽  
Resistance Mechanisms ◽  
Replication Forks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Genome Wide ◽  
Topoisomerase Poison ◽  
Recombination Repair ◽  
Non Homologous End Joining

SummaryMutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.One Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

scholarly journals Alzheimer’s disease-relevant tau modifications selectively impact neurodegeneration and mitophagy in a novel C. elegans single-copy transgenic model

2020 ◽  
Author(s):  
Sanjib Guha ◽  
Sarah Fischer ◽  
Gail VW Johnson ◽  
Keith Nehrke
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Genetic Model ◽  
Neurodegenerative Disorder ◽  
Model Organism ◽  
Single Copy ◽  
Mitochondrial Stress ◽  
C Elegans ◽  
Touch Receptor Neurons ◽  
New Perspective

ABSTRACTBackgroundA defining pathological hallmark of the progressive neurodegenerative disorder Alzheimer’s disease (AD) is the accumulation of misfolded tau with abnormal post-translational modifications (PTMs). These include phosphorylation at Threonine 231 (T231) and acetylation at Lysine 274 (K274) and at Lysine 281 (K281). Although tau is recognized to play a central role in pathogenesis of AD, the precise mechanisms by which these abnormal PTMs contribute to the neural toxicity of tau is unclear.MethodsHuman 0N4R tau (wild type) was expressed in touch receptor neurons of the genetic model organism C. elegans through single-copy gene insertion. Defined mutations were then introduced into the single-copy tau transgene through CRISPR-Cas9 genome editing. These mutations included T231E and T231A, to mimic phosphorylation and phospho-ablation of a commonly observed pathological epitope, respectively, and K274/281Q, to mimic disease-associated lysine acetylation. Stereotypical touch response assays were used to assess behavioral defects in the transgenic strains as a function of age, and genetically-encoded fluorescent biosensors were used to measure the morphological dynamics and turnover of touch neuron mitochondria.ResultsUnlike existing tau overexpression models, C. elegans single-copy expression of tau did not elicit overt pathological phenotypes at baseline. However, strains expressing disease associated PTM-mimetics (T231E and K274/281Q) exhibited reduced touch sensation and morphological abnormalities that increased with age. In addition, the PTM-mimetic mutants lacked the ability to engage mitophagy in response to mitochondrial stress.ConclusionsLimiting the expression of tau results in a genetic model where pathological modifications and age result in evolving phenotypes, which may more closely resemble the normal progression of AD. The finding that disease-associated PTMs suppress compensatory responses to mitochondrial stress provides a new perspective into the pathogenic mechanisms underlying AD.

scholarly journals Single-nucleotide resolution dynamic repair maps of UV damage in Saccharomyces cerevisiae genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3408-E3415 ◽  
Author(s):  
Wentao Li ◽  
Ogun Adebali ◽  
Yanyan Yang ◽  
Christopher P. Selby ◽  
Aziz Sancar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Model Organism ◽  
Early Time ◽  
Uv Damage ◽  
Single Nucleotide ◽  
Pyrimidine Dimers ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine–pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13–18 nucleotides 5′ and 6–7 nucleotides 3′ to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.

scholarly journals Characterization of the genome of the mealybug Planococcus lilacinus, a model organism for studying whole-chromosome imprinting and inactivation

2002 ◽  
Vol 79 (2) ◽  
pp. 111-118 ◽  
Author(s):  
K. NAGA MOHAN ◽  
PARAMITA RAY ◽  
H. SHARAT CHANDRA
Keyword(s):  
Drosophila Melanogaster ◽  
Repetitive Sequences ◽  
Gc Content ◽  
Model Organism ◽  
Fruit Fly ◽  
Single Copy ◽  
Coding Sequences ◽  
Repeat Sequences ◽  
Chromosome Imprinting

The co-occurrence of three chromosome-wide phenomena – imprinting, facultative heterochromatization and diffuse centromere – in the mealybug Planococcus lilacinus makes investigation of the genomics of this species an attractive prospect. In order to estimate the complexity of the genome of this species, 300 random stretches of its DNA, constituting ∼0·1% of the genome, were sequenced. Coding sequences appear to constitute ∼53·5%, repeat sequences ∼44·5% and non-coding single-copy sequences ∼2% of the genome. The proportion of repetitive sequences in the mealybug is higher than that in the fruit fly Drosophila melanogaster (∼30%). The mealybug genome (∼220 Mb) is about 1·3 times the size of the fly genome (∼165 Mb) and its GC content (∼35%) less than that of the fly genome (∼40%). The relative abundance of various dinucleotides, as analysed by the method of Gentles and Karlin, shows that the dinucleotide signatures of the two species are moderately similar and that in the mealybug there is neither over-representation nor under-representation of any dinucleotide.

scholarly journals Experimental Study on the Biological Effect of Cluster Ion Beams in Bacillus subtilis Spores

2019 ◽  
Vol 3 (2) ◽  
pp. 8 ◽  
Author(s):  
Yoshihiro Hase ◽  
Katsuya Satoh ◽  
Atsuya Chiba ◽  
Yoshimi Hirano ◽  
Shigeo Tomita ◽  
...  
Keyword(s):  
Biological Effect ◽  
Biological Effects ◽  
Model Organism ◽  
Lethal Effect ◽  
Molecular Ions ◽  
Ion Beams ◽  
Cluster Ions ◽  
Significant Difference ◽  
Cluster Ion Beams ◽  
Cluster Ion

Cluster ion beams have unique features in energy deposition, but their biological effects are yet to be examined. In this study, we employed bacterial spores as a model organism, established an irradiation method, and examined the lethal effect of 2 MeV C, 4 MeV C2, and 6 MeV C3 ion beams. The lethal effect per particle (per number of molecular ions) was not significantly different between cluster and monomer ion beams. The relative biological effectiveness and inactivation cross section as a function of linear energy transfer (LET) suggested that the single atoms of 2 MeV C deposited enough energy to kill the spores, and, therefore, there was no significant difference between the cluster and monomer ion beams in the cell killing effect under this experimental condition. We also considered the behavior of the atoms of cluster ions in the spores after the dissociation of cluster ions into monomer ions by losing bonding electrons through inelastic collisions with atoms on the surface. To the best of our knowledge, this is the first report to provide a basis for examining the biological effect of cluster ions.

Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis

Genome ◽  
2020 ◽  
Vol 63 (2) ◽  
pp. 61-90
Author(s):  
Vidhya Balasubramanian ◽  
Bharath Srinivasan
Keyword(s):  
Intracellular Calcium ◽  
Calcium Binding ◽  
Model Organism ◽  
Cell Types ◽  
Single Copy ◽  
Structural Determinants ◽  
Α Subunit ◽  
Behavioral Abnormalities ◽  
Mutant Phenotypes ◽  
Trisphosphate Receptor

Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have never been phenotypically characterized in any model organism. We have analyzed an adult-viable mutant that completely disrupts the G protein α-subunit binding and activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological/developmental, or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells variously rescues pleiotropic mutant phenotypes arising from impaired inositol 1,4,5-trisphosphate receptor (IP3R) activity (in turn depleting cytoplasmic Ca2+ levels across diverse tissue types). Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of amyotrophic lateral sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.

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