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[
Parasitol Int,
2014]
Little is known about the genetic variability of the soil-transmitted nematode, Strongyloides stercoralis, in humans. We sequenced portions of the small subunit rDNA (SSU), including the hyper variable regions (HVR) I and IV from S. stercoralis larvae derived from individuals living in a rural setting in Cambodia. We identified three polymorphic positions, including a previously reported one within the HVR I. HVR IV was invariable. Six different SSU alleles existed in our sample. Although different genotypes of S. stercoralis were found in the same individuals, no heterozygous larvae were found. This indicates that there is no or very little interbreeding between the different genotypes. Further studies are needed to examine if this is because sexual reproduction, which is facultative, is rare in our study area's S. stercoralis population or because what is considered to be S. stercoralis today is actually a complex of closely related species or subspecies.
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[
G3 (Bethesda),
2019]
Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of <i>S. cerevisiae</i> and <i>C. elegans</i> strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.
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[
Ecol Evol,
2014]
Organisms live in heterogeneous environments, so strategies that maximze fitness in such environments will evolve. Variation in traits is important because it is the raw material on which natural selection acts during evolution. Phenotypic variation is usually thought to be due to genetic variation and/or environmentally induced effects. Therefore, genetically identical individuals in a constant environment should have invariant traits. Clearly, genetically identical individuals do differ phenotypically, usually thought to be due to stochastic processes. It is now becoming clear, especially from studies of unicellular species, that phenotypic variance among genetically identical individuals in a constant environment can be genetically controlled and that therefore, in principle, this can be subject to selection. However, there has been little investigation of these phenomena in multicellular species. Here, we have studied the mean lifetime fecundity (thus a trait likely to be relevant to reproductive success), and variance in lifetime fecundity, in recently-wild isolates of the model nematode Caenorhabditis elegans. We found that these genotypes differed in their variance in lifetime fecundity: some had high variance in fecundity, others very low variance. We find that this variance in lifetime fecundity was negatively related to the mean lifetime fecundity of the lines, and that the variance of the lines was positively correlated between environments. We suggest that the variance in lifetime fecundity may be a bet-hedging strategy used by this species.
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[
Mol Ecol,
2013]
Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools.
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[
East Coast Worm Meeting,
2004]
Targeting Induced Local Lesions In Genomes, or TILLING R , is a novel high throughput technology for reverse genetics. TILLING uses chemical mutagenesis to yield a traditional allelic series of point mutations for virtually all genes. TILLING is of particular value for essential genes where sublethal alleles are required for phenotypic analysis. TILLING has become an established technique on many model organisms, including C. Elegans, Arabidopsis, Zebrafish, Maize, Rice, and others. A variation on TILLING is called Ecotilling, in which high throughput SNP discovery is performed by locating natural variations throughout the genome. LI-COR Biosciences is a manufacturer of DNA Analysis Instrumentation for high throughput TILLING and Ecotilling, as well as for DNA Sequencing, AFLP R , and Microsatellite analysis. LI-COR also manufactures instrumentation for infrared fluorescent protein imaging, offering superior quantification over standard chemiluminescence. Stop by our booth to pick up the latest literature and publications on our products. TILLING is a registered trademark of Anawah, Inc. AFLP is a registered trademark of KeyGene, N.V.
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[
Sci Rep,
2015]
Dietary restriction appears to act as a general non-genetic mechanism that can robustly prolong lifespan. There have however been reports in many systems of cases where restricted food intake either shortens, or does not affect, lifespan. Here we analyze lifespan and the effect of food restriction via deprived peptone levels on lifespan in wild isolates and introgression lines (ILs) of the nematode Caenorhabditis elegans. These analyses identify genetic variation in lifespan, in the effect of this variation in diet on lifespan and also in the likelihood of maternal, matricidal, hatching. Importantly, in the wild isolates and the ILs, we identify genotypes in which peptone deprivation mediated dietary restriction reduces lifespan. We also identify, in recombinant inbred lines, a locus that affects maternal hatching, a phenotype closely linked to dietary restriction in C. elegans. These results indicate that peptone deprivation mediated dietary restriction affects lifespan in C. elegans in a genotype-dependent manner, reducing lifespan in some genotypes. This may operate by a mechanism similar to dietary restriction.
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[
Mol Syst Biol,
2012]
The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C. elegans strains (genotype) cultivated in five growth conditions (environment), and find that highly regulated genes, as distinguished by intergenic lengths, motif concentration, and expression levels, are particularly biased toward genotype-environment interactions. Sequencing these strains, we find that genes with expression variation across genotypes are enriched for promoter single-nucleotide polymorphisms (SNPs), as expected. However, genes with genotype-environment interactions do not significantly differ from background in terms of their promoter SNPs. Collectively, these results indicate that the highly regulated nature of particular genes predispose them for exhibiting genotype-environment interaction as a consequence of changes to upstream regulators. This observation may provide a deeper understanding into the origin of the extraordinary gene expression diversity present in even closely related species.
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Kumar, J., Vargas, M., Kapahi, P., Katewa, S., Khanna, A., McCloskey, T., Gill, M.
[
International Worm Meeting,
2015]
During development C.elegans can enter an alternate larval stage called the dauer larva, which allows it to survive adverse environmental conditions. Food is one of the factors that influences whether an arrested dauer will resume reproductive growth. The dauer development decision occurs during molting from L1 to L2 larval stage, and undergoes an underlying metabolic shift controlled by food signal. Embryos and L1 larvae use the glyoxylate cycle to generate carbohydrate from lipid stores. In L2 and later stages animals perform aerobic respiration and increase TCA cycle activity. In contrast, dauer animals do not perform aerobic respiration. Surprisingly, little is known about the chemical nature of the food signal(s), although the molecular pathways through which they signal have been partially defined. Molecular genetic analysis of dauer-constitutive (daf-c) and dauer-defective (daf-d) mutants show that dauer formation is regulated by complex interactions between at least three major signaling pathways: a guanylyl cyclase pathway, a TGF-beta like pathway and an insulin-like pathway. Food activates the guanylyl cyclase DAF-11 and promotes transcription of the TGF-beta ligand DAF-7 as well as activating the insulin-signaling pathway. The insulin-like signaling pathway (ILS) responds to dietary cues by moderating levels of genes involved in dauer formation, stress response, and lifespan regulation through its downstream transcription factor, DAF-16/FOXO. This suggests that E. coli is not simply a source of calories but also provides critical signals that are required for full C. elegans nutrition and development, suggesting a relationship that mimics microbiota function (Collino et al., 2013). Here we demonstrate how individual gene knockouts in E. coli can modify signaling pathways in C. elegans to influence development and survival. Through a systematic screen of ~4000 single gene knockout E. coli strains, we identified 56 E. coli mutants that not only enhance dauer formation in a C. elegans insulin-like receptor mutant,
daf-2, but also extend adult lifespan. We also demonstrate how genetic analysis can be used to determine the molecular mechanism by which the bacteria influence worm physiology by focusing on one bacterial mutant, cyaA (adenylate cyclase). Our results demonstrate how mutant bacterial strains can be used to study intricate questions concerning the relationship between dietary factors and bacterial products with the phenotypes of the consuming organism.
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Jelic M, Hsieh EJ, Pruett D, Kennedy BK, Ros V, Murakami CJ, Xian B, Carr D, Han JD, Pradeep P, Johnson SC, Moller RM, MacCoss MJ, Wende HV, Tocchi A, Holmberg M, Klum S, Chen W, Delaney JR, Olsen B, Goswami S, Lo W, Spector BL, Liao E, Peng ZJ, Pollard T, Rai D, Castanza A, Wasko BM, Fletcher M, Bennett CF, Yu T, Singh M, Jeong KS, Lin MS, Kim JR, Rabinovitch PS, Kaeberlein M, Miller H, Schleit J, An EH, Simko M, Sutphin GL, Higgins S, Trongtham N
[
Aging Cell,
2013]
Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.
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[
Microb Pathog,
2020]
Most of the human gene homologs are found in Caenorhabditis elegans. As a wide variety of micro-organisms present in the environment is pathogens, so, C. elegans could be a useful model to track future infectious disease. With this knowledge, in this study, we isolated Acinetobacter courvalinii from the soil and characterized its pathogenicity for the first time. For the isolation, we used Glucose-Yeast extract-Ethanol-Calcium carbonate medium. To this aim, we evaluated the resistivity of bacteria against several stressful microenvironments. As we observed, A. courvalinii JP_A1001 shown highly tolerance against the acid environment (pH 3-7), resistant against up to 0.2% of phenol content, and survived in the medium supplemented with 0.3% of bile salt. In addition, the bacteria were also resistant against several antibiotics showing the property of multidrug-resistant bacteria. Moreover, the isolated bacteria have shown the biofilm formation ability within 60h. Further, we found that incubation of C. elegance with A. courvalinii JP_A1001 decreased the body movement and increased the free radical generation which remarkably influenced the life expectancy of C. elegans compared to E. coli OP50. Therefore, we concluded that A. courvalinii JP_A1001 found in the soil could be a future threat as a pathogen to public health.