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[
Ann Bot,
2003]
Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15% below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15% more DNA than 2C chicken nuclei (with gtoreq2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75% above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57% above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.
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[
PLoS Genet,
2013]
Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [
mus-81;
xpf-1], [
slx-1;
xpf-1], [
mus-81;
him-6] and [
slx-1;
him-6] double mutants showed a similar reduction in survival rates as
slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.
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[
International Worm Meeting,
2003]
We have identified two C. elegans proteins containing ERCC4-like domains, which are responsible for incising damaged DNA: the XPF orthologue (C47D12.8) and a gene that we have named
mus-81 (C43E11.2) because it appears to be an orthologue of yeast Mus81. Yeast Mus81 (MMS and UV sensitive 81) was identified through 2-hybrid screens; in S. pombe Mus81 interacts with Cds1, a cell cycle checkpoint kinase, and in S. cerevisiae Mus81 interacts with Rad54, a DNA repair protein. Mus81 is reported to participate in repair of DNA damage caused by UV irradiation or MMS. The
mus81 orthologue in C. elegans is likely to be the terminal gene in an operon. We have shown that a 1.3 kb
mus-81 transcript is produced and trans-spliced to SL2. All known Mus81 proteins contain two non-sequence specific DNA-binding hairpin-helix-hairpin motifs flanking the ERCC4 domain. The predicted C. elegans MUS-81 is no exception and shares 50% similarity to Mus81 proteins from other phyla. Depletion of
mus-81 by RNAi causes hypersensitivity to UV irradiation; treated animals display a decrease in brood size, a reduction in viability and an increase in germ cell apoptosis when compared to similarly treated N2 worms. In addition, continuous depletion of
mus-81 by RNAi through multiple generations eventually leads to sterility and an increased frequency of abnormal karyotypes. Together these results suggest that
mus-81 plays a role in maintaining genome stability. Yeast that lack Mus81 are defective in meiosis and in vitro analysis suggests that Mus81 might be a Holliday Junction (HJ) resolvase. We have failed to detect meiotic defects in
mus-81 (RNAi) worms, so it is unlikely that MUS-81 functions as a HJ resolvase in C. elegans. We are investigating the possibility that the C. elegans XPF endonuclease orthologue is the HJ resolvase. Presently we are analysing the expression patterns of
mus-81 gene products by Northern analysis and through the use of a MUS-81::GFP reporter. The fusion is expressed in the nucleus and is enriched in the nucleolus. We are also pursuing protein interaction studies to identify MUS-81 partners and have generated an anti-MUS-81 polyclonal antibody to aid in our analysis.
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[
MicroPubl Biol,
2024]
High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of <i>Caenorhabditis elegans</i> research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hands-on time and sustainability compared to current standardized DNA extraction protocols, while equally effective. This work indicates that it is useful to compare methods from different model systems, such as mammalian organoids and invertebrate nematodes, to find useful alternatives for research methodologies.
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[
International Worm Meeting,
2015]
Investigation of the neuronal basis of economic decisions would be accelerated by establishing decision making paradigms in simple, genetically tractable organisms, such as the nematode Caenorhabditis elegans. For an organism to be a valid model of economic decision making its choice behavior must be sensitive to: (i) the difference between high and low quality goods, and (ii) the relative cost of those options.Previous work has shown that the nematode worm C. elegans quickly learns to feed on those foods (species of bacteria) that promote higher rates of growth and reproduction. Worms spend more time foraging in patches of Good bacteria (high worm growth rate) versus Mediocre bacteria (moderate growth rate) when equally abundant. Until now, however, it has not been possible to simultaneously present two food choices of different quality and cost. To that end, we have developed an electro-microfluidic device in which a semi-restrained worm forages between contiguous yet discrete fluid streams containing good and mediocre quality food. This arrangement constitutes a two-alternative forced-choice task, analogous to those used in behavioral economics. Electrodes inserted into the device monitor muscular impulses associated with individual swallowing events. Relative consumption of Good and Mediocre food is measured by counting the number of swallowing events in the respective fluid streams. The fraction of total swallowing events in Good vs Mediocre food serves as an index of food preference. Importantly, we can alter the effective prices of the two foods by adjusting the concentration of the bacteria, with price being inversely related to concentration.Here we present behavioral data delineating preference for Good vs Mediocre food across a range of relative prices. We find that worms exposed to the two species of bacteria at equal prices prefer Good bacteria, indicating that feeding preferences are normal in the device. Worms respond to price adjustments as predicted by economic theory in that increasing the relative price of a food leads to a decline in its consumption. In addition, we present calcium-imaging data from sensory neurons showing that they respond to transitions between Good and Mediocre foods, and the amplitude of calcium signal scales with relative food preference. These results show that C. elegans forages in an economic manner, and that relative value is represented at the level of the sensory neurons.
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[
International Worm Meeting,
2017]
Value-based decision making - choices driven by subjective assessments of utility - is a central function of the brain and the focus of intensive study in mammals. Until now, evidence that nematodes are capable of value-based decision making has mainly been suggestive. However, economists have developed formal procedures for determining whether a consumer's decisions are based on subjective value as opposed to random or capricious impulses. We recently developed microfluidic devices that enable such tests to be performed on nematodes for the first time. The worm is held at the confluence of contiguous streams of high and low quality bacterial food leaving its head free to move. Bacteria concentrations are adjusted by the experimenter to change the relative "prices" of the two foods in terms of number of bacteria consumed per pharyngeal pump. Food concentrations can also be adjusted in tandem to increase or decrease the worm's overall consumption possibilities, i.e. "budget." Consumption is measured by counting pharyngeal pumps recorded electrically. Worms typically fed in both streams, consuming a mixture of high and low quality food that was unique for each combination of price and budget. We found that worms make globally rational choices in that they obey transitivity. That is, for all sets of food mixtures A, B, and C, if A is preferred to B, and B to C, then A is preferred to C. As transitivity is the necessary and sufficient condition for value maximization, these data provide formal evidence that C. elegans exhibits value-based decision making. Further, we found that the olfactory neuron AWC, known to be activated by the sudden absence of food, is required for intact food choice behavior. Surprisingly, however, we found that AWC is also activated by the switch from high quality food to low quality food, even when the two foods are at the same concentration (price). Thus, subjective value may be represented at the level of individual olfactory neurons. Our behavioral and neuronal data are consistent with a model in which olfactory neurons represent the subjective value of the local environment to direct behavior toward preferable mixtures of particular foods. To our knowledge, this is the first formal demonstration of value-based decision making in a genetically tractable model organism with a simple nervous system, opening the door to the discovery of conserved genes and neural circuits for rational decision making.
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[
International Worm Meeting,
2009]
Behavioral and developmental choices made by any animal represent a strategy for surviving and reproducing in its environment. The essence of strategy is making decisions on the basis of incomplete information, decisions whose costs and benefits can''t be accurately determined at the time they must be made. Worms make many such choices: the decision to leave low-quality food in search of higher quality, the decision to lay eggs or allow them to hatch internally, and the decision to become a dauer or remain a dauer are examples. Not coincidentally, most of these decisions involve food availability, perhaps the most important environmental variable to a worm. I am trying to quantitatively model such decisions, using mathematical tools developed for financial markets. The L2/L2d decision is particularly interesting, because it appears to be unnecessary. An L2d can become either an L3 or a dauer, while an L2 can only become an L3. Since the L2d can do everything the L2 can do and more, why does the L2 exist? A likely answer is suggested by the work of Golden and Riddle. They showed that L1 L2d L3 pathway takes a few hours more than L1 L2 L3. Under ideal conditions a worm population doubles in 10-11 hours. (This number is calculated from published life-history traits, and is in approximate accord with lab experience.) A delay of 7 hours, therefore, reduces fitness by a factor of 27/10.5 = 0.62. Thus a worm that becomes an L2d pays a price of about 40% of its fitness for the option of eventually becoming a dauer. A worm should become an L2d if it can confidently predict that conditions will be so bad in the future as to cause a decrease of fitness of this magnitude. Most of what we know about dauer formation concerns how the worm evaluates environmental conditions. However, the L2d should also be preferred in highly uncertain environments, since it postpones the dauer decision into the future, when more accurate information will be available. This effect can be modeled using the tools of stochastic calculus, used to price options in financial markets. They predict that the L2/L2d decision should be strongly influenced not only by how good the environment is, but also by how volatile it is.
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[
Worm Breeder's Gazette,
1992]
After tabulating the results of the Worm Plate Survey. we have come up with some interesting results. Most notably. the high variability in prices that labs are paying for their plates, even for the exact same plates from the same supplier, and the fact that most plates are marked up considerably over the actual cost. The replies can be separated into 4 categories: Labs that get plates from Fisher ($29-$58). but wish they had non-vented plates Labs that get non-vented plates via Applied Scientific (~$38) Labs that get plates from Falcon (vented) or Nunc (non-vented) and pay much more Most labs' plates were "slipable" or "semi-stackable", but all labs wanted plates that stack well for easy manual pouring, seeding, carrying, and using. Everyone wanted plates with shallow lids such that the bottoms can be lifted out of the tops for inverted use. Some labs expressed an interest in plates slightly smaller than "60 mm". That number is in quotes because all of the companies' plates have bottoms smaller than 60 mm (e.g. Fisher -54 x 14 mm). We have negotiated with the plastic companies that really make the plates for Fisher, Applied Scientific, etc. (that actually just resell them to you). I have come to the conclusion that we can provide you with better worm plates, the same worm plates cheaper, or in most cases better worm plates cheaper. This is true for every lab. The bottom line is that we can get you top quality non-vented "60 mm" plates (like Applied Scientific's, except fully stackable) for about $29 per 500 case INCLUDING shipping depending on your usage and how many cases you can receive at one time. Several labs have found the non-vented plates last longer without drying out or getting contaminated, compared with normal vented plates, so you should save that way, too. We offer full service shipping (e.g. standing orders and same-day telephone orders, free. Similarly low prices are available on 100 mm and 150 mm plates that exceed industry standards for flatness (reducing media usage) and clarity. The 100 mm are about $27 per 500 case plus shipping; The 150 mm dishes (good for DNA & RNA preps and library platings, with more than 2.25x the surface area of 100 mm dishes) are made thicker and deeper than industry standards and are about $21.50 per 100 case plus shipping. The shipping charge is very low for labs, or groups of labs in one city, that can take delivery of many cases in a single shipment. You can even suggest that your stockroom order plates from us. Call us for an exact price quote depending on your usage and how many cases you can receive at one time. In any case, we'll work things out to save you money. In the future, we can offer inexpensive 35 mm dishes if the community at large can order about 2000 cases per year, so let me know about your needs for other sizes. The response was very mixed about pre-poured plates. We may set that up later, but for now we can help the most by saving you lots on empty petri dishes (and later, maybe media .supplies). We are happy to send out free samples so you can examine the dishes. If we haven't contacted you yet, just give us a call. Respondents: 38 (including 5 anonymous) "Winners": Horvitz = 550, Meyer = 400, Thomas = 400, Greenwald = 300 200-299 cases 8 labs 100-199 cases 7 labs 4-99 cases 19 labs Highest price per case: US = 118.75, Canada = $117 (non-vented) Lowest price per case: US = $29, Canada = $25 (vented) Farthest away response: Malta! No responses from MRC or anyone else in Europe or Asia. It is possible that we can save money and/or provide better plates for these labs, including, shipping, too. Let us know.
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Filippidis, G, Papazoglou, TG, Voglis, G, Kapsokalyvas, D, Tavernarakis, N, Kouloumentas, C
[
J Phys D Appl Phys,
2005]
Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing ferntosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.
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[
PLoS Genet,
2010]
DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.