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[
Worm Breeder's Gazette,
1995]
An NGM plate full of worms and bacteria forms a complete two-species microcosm that can be useful for studies of ecological dynamics. I wasled to this project while trying to develop a suitable discrete generationprotocol for artificial selection studies. Cleaning plates with asodiumhypochlorite solution every four days (20C) produces sequentialpopulations without having to worry about overlapping generations or dauerformation. While working out this method, however, I noticed that in somegenerations there would be tens of thousands of worms on a plate whereasother generations would yield only a few thousand worms. The functionalexplanation for this turned out to be density dependent populationregulation. When there are a few worms on a plate, there is plenty offood and many eggs are produced for the next generation (the populationincreases). When the number of worms becomes large, however, worms begindeveloping more slowly, and may not be producing eggs by day four. This can lead to a crash in population size. Ordinarily, we would expect asteady state to be reached at the carrying capacity of the plate. C.elegans produces an extremely large number of offspring, however, and itsnet reproductive rates is large enough to theoretically generate complexpatterns of population dynamics, including chaos. To test this hypothesis, I raised four replicate lineages of N2worms for 100 generations using the serial transfer technique describedabove (10cm plates). The the volume of OP50 inoculum was controlled,and worm densities were estimated using serial dilutions of the wormsrinsed off the plates. Although the population dynamics during this timewere indeed complex (see figure--record high 78,900; carrying capacityprobably around 15,000), the fact that replicates occasionally tend tocycle in concert suggests that environmental factors as well as populationregulation effects are influencing population dynamics. Possibleenvironmental influences are small variations in the time betweencleanings, plate age and quality, and E. coli culture age and quality. The data still need to be analyzed using more sophisticated methods before can I determine whether a chaotic signature emerges from the environmental noise, but one message I take from this is that if it is difficult to separate these features in a tightly controlled system suchas C. elegans plates, then doing it in natural populations might well beimpossible.
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[
Trends Genet,
2006]
Recent studies show that local populations of the nematode Caenorhabditis elegans possess nearly as much genetic variation as that seen in existing worldwide collections. This suggests either wide-ranging migration and intense natural selection or recent dispersal, perhaps by human association. Either way, the effective population size of this ubiquitous model organism is unexpectedly small.
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[
Nat Genet,
2012]
A new study reports a comprehensive survey of genetic diversity in natural populations of the nematode Caenorhabditis elegans. Their analyses suggest that recent chromosome-scale selective sweeps have reduced C. elegans genetic diversity worldwide and strongly structured genetic variation across its genome.
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[
International Worm Meeting,
2011]
Small RNA mediated silencing of transposons is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the exogenous and endogenous small RNA pathways genes have been demonstrated to be involved in transposon silencing. When these genes are mutated, transposons can be excised, leaving a double-strand break, which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining.
I have developed a strategy to visualize double-strand breaks in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to look at transposon hopping in mutants that are sterile and possibly to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the small RNA pathways and genome integrity, and further elucidate the mechanisms of transposon silencing in the germline.
CMP is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation.
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[
Methods Mol Biol,
2009]
The nematode Caenorhabditis elegans has emerged as an informative experimental system for analysis of meiosis, in large part because of the advantageous physical organization of meiotic nuclei as a gradient of stages within the germline. Here we provide tools for detailed observational studies of cells within the worm gonad, including techniques for light and electron microscopy.
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[
Genetics,
2022]
The transparency of Caenorhabditis elegans provides a unique window to observe and study the function of germ granules. Germ granules are specialized ribonucleoprotein (RNP) assemblies specific to the germline cytoplasm, and they are largely conserved across Metazoa. Within the germline cytoplasm, they are positioned to regulate mRNA abundance, translation, small RNA production, and cytoplasmic inheritance to help specify and maintain germline identity across generations. Here we provide an overview of germ granules and focus on the significance of more recent observations that describe how they further demix into sub-granules, each with unique compositions and functions.
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[
International Worm Meeting,
2009]
Small RNA-mediated gene silencing is a conserved mechanism used by nearly all eukaryotes as a way to regulate gene expression at both the transcriptional and post-transcriptional level. The double-strand RNA can be delivered from outside the cell or can be generated from endogenous transcription of coding and non-coding genomic loci. Some processes known to be regulated by RNAi include transposon silencing, defense against viral infection, and maintenance of heterochromatin. Transposon silencing is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the genes shown to be involved in exogenous RNAi have also been shown to be involved in transposon silencing in the germline, including
mut-2,
mut-7,
mut-14,
mut-15,
mut-16, and
rde-2. When these genes are mutated, transposons can be excised, leaving a double-strand break (DSBs), which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining. I am currently developing a strategy to visualize DSBs in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. The tagged constructs that I am generating for cytology will include a modified version of the Tandem Affinity Purification (TAP) tag generated by Cheesman et al. (2004), which includes a fluorescent tag followed by a TEV protease cleavage site and the S peptide domain and is referred to as the Localization and Affinity Purification (LAP) tag. It allows for both visualization of the protein and its affinity purification. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the RNAi pathway and the maintenance of genome integrity, and further elucidate the mechanisms of transposon silencing in the germline.
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[
International C. elegans Meeting,
1995]
C. elegans populations raised via serial transfer with discrete generations display fluctuations in population size that can span several orders of magnitude. This is somewhat unusual since the worms are maintained under controlled conditions on a fixed amount of resource (OP50). Large fluctuations in population size in an apparently constant environment could be the signature of deterministic chaos induced by density dependent population size regulation and the very large reproductive capacity of C. elegans. To test this hypothesis, I raised four replicate lines of worms for 100 generations at 20 C, transferring the populations every four days using the hypochlorite cleaning procedure (i.e., only eggs were transferred). Analysis of the resulting time series shows that there is strong density dependent regulation in these populations, but that the size fluctuations are driven mostly by environmental variation rather than deterministic chaos. The environmental influences are probably caused by variation in the age and quality of E. coli cultures and plates, as well as small differences in the timing of transfers. Large scale sensitivity resulting from small amounts of input variation probably arises from "local chaos" during different parts of the population regulation cycle. This study demonstrates that C. elegans can be developed into a useful model system for ecological and evolutionary studies.
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[
International Worm Meeting,
2015]
The Caenorhabditis Intervention Testing Program (CITP) is a multi-institution effort, sponsored by the National Institute of Aging, designed to screen bioactive compounds for their ability to extend lifespan and enhance health using nematodes as a model system for the potential effects of background genetic variation. The Intervention Testing Program (ITP), began assaying pharmacological interventions for lifespan extension in mice in the early 2000s [1]. However, with a median lifespan over two years, this project has decreased capacity to test multiple compounds in a robust manner [2]. The CITP capitalizes on the short lifespan of hermaphroditic C. elegans, C. briggsae and C. tropicalis. Selected natural isolates represent a biologically and geographically diverse population, yielding a total of 22 test strains. A compound effective across a diverse worm panel increases the likelihood of potency across other species, including heterogeneous human populations.Reproducibility marks a key goal of the CITP; the collaborative group generated detailed standardized protocols to minimize among and within lab variability. A data center facilitates near-instantaneous and global access to all CITP data. Compound candidates are selected based on the scientific community's recommendations and advice from the project's steering committee.A subset of strains has already undergone initial testing of four compound interventions: (R) alpha-lipoic acid, Aspirin, Quercetin, and NP1. Resveratrol, propyl gallate, and Thioflavine T are to be tested shortly. In addition to manual lifespan experiments, each lab built over 20 automated lifespan machines which use flatbed scanner technology [3], allowing for high throughput and precise longevity measurements. Mid- and late-age health assays are also underway.Initial results indicate significant differences in among-isolate variation in longevity within species (e.g., C. elegans low variation, C. briggsae high variation), as well as strain- and species-specific differences in longevity responses to compound treatments.1. Warner et al (2000) Mech Ageing Dev 115:199-2072. Miller et al (2007) Aging Cell 6:565-5753. Stroustrup et al (2013) Nature Methods, 10:665-670.
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[
Curr Biol,
2015]
A new study investigating the origins of diversity in the structure of the mitotic spindle in nematode embryos, at timescales spanning a few generations to hundreds of millions of years, finds that most features of the spindle evolve viaascaling relationship generated by natural selection acting directly upon embryo size.