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[
Worm Breeder's Gazette,
1994]
FROM ASCARIS TO C. ELEGANS: A WAY TO STUDY GENE STRUCTURE AND FUNCTION Huang Y-J., Tobler H. and Muller F., Institute of Zoology, University of Pribourg, Perolles, CH-1700 Fribourg, Switzerland
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[
Worm Breeder's Gazette,
1998]
Correction: "The abstract entitled Control of Mitochondrial Morphology published in WBG Vol. 15 No. 1 contained an error. Worms were treated with 3 mM (not 100 mM) chloramphenicol to induce changes in mitochondrial morphology. Thanks to A.L. for spotting the error. M. Crawley & D. Adams
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[
Worm Breeder's Gazette,
1975]
Using Ascaris as a model system, we are studying ionic mechanisms of the spontaneous electrical activity in nematode somatic muscle. The fast spike potentials appear to be Ca+2 mediated; their amplitude depends on the external Ca+2 concentration, they are TTX insensitive, they persist when Na+ is replaced by Tris+, choline+, or Cs+, and they are blocked by Co+2 and La+3. When the normal solution is replaced by one containing 11 mM Ba+2 and 0.15 mM Ca+2 as the only divalent cations, the slow waves underlying the normal spike activity appear to increase dramatically in amplitude and duration; spike activity gradually disappears. The duration and amplitude of the slow waves at steady state under these conditions increase with Ba+2 concentration, reaching values of 1-2 minutes and 50-60 mV, respectively, in 26 mM Ba+2. These results and others lead us to conclude that the slow waves are also Ca+2 mediated. The muscles are depolarized by 0.1 mM ouabain, suggesting some involvement of an electrogenic pump in maintaining the membrane potential. TEA, in concentrations as low as 1 mM, has pronounced effects on the spontaneous myogenic activity, consistent with the effects observed when TEA is injected iontophoretically into C. elegans.
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[
Worm Breeder's Gazette,
1992]
We have developed a method to histochemically stain C. elegans that does not kill the embryos inside the stained mother and we have used this method to isolate mutations that alter gene expression patterns (see the accompanying article by Guofeng Xie et al. on the selection of mutants based on histochemical staining patterns). To make C. elegans permeable to the histochemical stains, we modified Finney and Ruvkin's method to make C. elegans permeable for antibody staining. We have stained animals permeabilized in this way for esterase, -glucuronidase and , -galactosidase. This procedure should work for other histochemical stains as well. In our hands, worms made permeable by this method and stained for , -galactosidase activity are better stained than those made permeable by the freeze-dry method and stained for , -galactosidase. Essentially all animals taken en mass through this procedure are stained. Approximately 75% of the embryos inside the stained animals survived and there were an average of 14 + 8 (n= 17) surviving offspring from each stained mother. Our protocol varies slightly depending on whether we are staining for esterase or -galactosidase. We present the -galactosidase protocol here. 1. Formaldehyde preparation: mix 200 mg of paraformaldehyde with 900 l of 5.0 mM NaOH. Heat the solution at 65 C for 15 min. Centrifuge this mixture in a microfuge at 14,000 rpm for 1 min. Use the supernatant immediately. 2. Wash healthy gravid worms from a 60 mm NGM plate with 1-2 ml of M9 and transfer the worms to a microfuge tube. 3. Let the worms settle for 5 min at room temperature and remove the supernatant. 4. Add 500 / l of ice-cold 2X MRWB (160 mM KCl, 40 mM NaCl, 20 mM Na2 EGTA,10 mM spermidine HCl, 30 mM PIPES, pH 7.4 and 50% methanol), 100 l of formaldehyde and 400 l of water and mix well. Incubate the tube for 35 min at 4 C. Occasionally mix the tube contents during fixation. 5. Wash 2X with 1.0 ml TTB (100 mM Tris HCl pH 7.4, 1% Triton X-100 and 1.0 mM EDTA). 6. Add 960 l of TTB, 30 l formaldehyde and 10 l of , -mercaptoethanol. 7. Place the tube on a rotator set at 2 rpm for 10 min at room temperature and then let the worms settle for 5 min at room temperature. At this point the worms are fragile so treat them carefully. 8. Wash once with 1X B03 (40X B03 contains 1.0 M H3B03 and 0.5 M NaOH) 9. Add 900 l of 1X B03 and 100 l of 100 mM DTT. 10. Mix on a rotator at 2 rpm for 15 min at room temperature. 11. Wash twice with ddH20 letting the worms settle for 5 min each time. 12. Stain for -galactosidase activity. We use a very slightly modified version of Andy Fire's staining mix: 620 l H20 ,250 l 0.8 M Na phosphate (pH 7.5), 2 l 1.0 M MgCl2 ,4 /ll 1% SDS, 100 l Redox buffer (50 mM each of Potassium Ferricyanide and Potassium Ferrocyanide), 5 l 5.0 mg/ml Kanamycin Sulfate and 20 l 2% X-GAL in Dimethylformamide. 13. We stain at room temperature for 8-10 hours (but the staining time will vary with the lacZ construct).
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[
Worm Breeder's Gazette,
1989]
At the meeting in May there was considerable interest in the protocol we have been using to obtain nuclear extracts. The procedure we use to obtain the nuclei is that described by Dixon et al (WBG 2 (3) :73-74) except that PMSF is present at 1 mM in all buffers and 1 mM DTT is used in place of mercaptoethanol. The efficiency depends in great part on the tightness of the homogenizer used to shear the worms. The nuclei are washed twice in 10 ml of TKM (50 mM Tris-HCI (7.5), 25 mM KCL and 5 mM MgCl2) and resuspended in 3 volumes of TKM. At this point the volume of nuclei is sufficiently small (0.05-0.2 ml) to carry out the remaining steps in microfuge tubes. EDTA is added to 0. 01 N and an equal volume of 2X Iysis buffer (1X= 40 mM Tris-HCI (7.5), 1 mM EDTA, 0.5 mM DTT, 1 M NaCl, 10% glycerol) is added. Tubes are mixed gently for 5 minutes in the cold and one-half volume of 18% PEG8000 in 1X Iysis buffer is added slowly with gentle mixing. The tubes are slowly rotated in the cold for 1 hour and nucleic acids are removed by centrifugation (17000 G for 20 minutes). This crude supernatant appears to be free of nucleases and proteases and can be used directly or dialyzed against your favorite buffer using micro dialysis with membranes having a 12 kD cut-off, protein concentration decreases by a factor of about 2 during dialysis. Two grams of worms yields 0.5 to 1 mg of protein before dialysis. The extracts have little contamination by cytoplasmic components as the vitellogenins appear to be absent when SDS-PAGE is performed. Using extracts from mixed stage and sex N2s, we have repeated the gel retardation experiments which we described at the meeting. Specific binding can be demonstrated with a double-stranded substrate containing the TATTGAAA sequence which we have suggested is an X- linked binding site used in X/A ratio assessment. This binding is competed by double-stranded oligos which contain the octamer but not by those which lack it. The negatives include a sequence which precisely deletes the 8 base pairs but leaves 56 nucleotides around the site intact. Single stranded oligonucleotides, including the two complementary strands annealed to give a strong specific competitor do not compete for binding. Curiously, the 8 bp sequence by itself does not compete even when ligated into a large concatemer despite the fact that it is feminizing in the microinjection assay which we have previously described (WBG 10 (1): 62-63).
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[
Worm Breeder's Gazette,
1977]
1. New Zeiss immersion oil (brown bottles) is toxic to C. elegans, whereas old oil (containing PCB, white bottles) is innocuous. New oil stops worms pumping immediately upon contact, while the PCB oil does not. L2s mounted on agar surrounded with new oil, fail to produce progeny. In the short term, this may not matter; indeed, the oil repels the worms and helps to stop them escaping. For long-term viability, 18 mm cover slips can be used so that the oil can be kept away from the agar: edges are sealed with Voltalef oil. J.E.K. mounts the worm on a 12 mm slab and fills in the edges with 2% agar. 2. Rapid demounting (a) Brief inspection. Trimming and sealing not necessary. (b) Stable mount. The worm is mounted under a 13 mm circular cover slip; the agar is not trimmed but covered with a square of Saran Wrap containing an 11 mm hole. Seal is good enough to prevent drying overnight. Very little immersion oil is applied to avoid wetting the plastic. Saran Wrap and cover slip can be removed quickly, leaving worm accessible on the agar (watch while lifting cover slip). Useful for:-laser microbeam operations, rapid fixation of transitory states. 3. Invertible mount - Horrible method: Developed for watching lateral cells through flips of later lethargi. 2% celloidin/amyl acetate spread on horizontal microscope slide (~0.1 ml/cm2); air dried under dust cover several days. 60 layer of 1% agarose cast on 22 x 40 mm cover slip (cellulose tape spacers); allowed to dry to ~40 ( edges just receeding); minute drop of buffer added with worm, covered with 10 mm square of the celloidin film, whose centre has been coated with bacteria as usual. Edges allowed to dry; flooded with immersion oil (new will do). Nomarski only slightly degraded through agarose; restraint of worm poor, dehydrates in a few hours. More details from J.E.S.
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[
Worm Breeder's Gazette,
1994]
The protein talin is found in a variety of vertebrate cells. In skeletal muscle it is located at myotendonous junctions and costamers. In non-muscle cells it is found at focal adhesions, sites where the actin cytoskeleton associates with the transmembrane protein integrin. It binds relatively weakly to the proteins integrin and actin, and relatively strongly to the protein vinculin in vitro. Its cellular location and biochemistry suggests that it is one link in a chain of proteins that function to attach actin to the membrane. The dense bodies found in C. elegans body wall muscle are sites where actin filaments are linked to the cell membrane. Dense bodies are known to contain the proteins integrin, vinculin, alpha-actinin and actin. They are, therefore, good models for the actin-membrane attachments found in vertebrate cells. We and others have been characterizing the proteins found in the dense body. Mutations exist that eliminate integrin and vinculin. Hresko et al. (M. Hresko, personal communication) have examined the organization of several muscle proteins in these mutants. Their data shows that integrin can assemble in the absence of vinculin, but that integrin is necessary for the localization of vinculin to the dense body. This data suggests a model in which the dense body assembles at the membrane beginning with integrin. We intend to examine how talin fits into this model. Michael Hengartner in the laboratory of R. Horvitz recovered a putative C. elegans talin cDNA in a search for
unc-69 cDNAs. The clone was subsequently found to be chimeric, and the talin homology unrelated to
unc-69 .This clone was kindly given to us for further analysis of the talin homologous region. It was hybridized to the YAC grids from the physical mapping project and found to map to the clone Y71G12 .This clone derives from the far left of LG I. There are no known muscle affecting genes in this part of the map. We have recovered additional, uncorrupted talin cDNAs. We have nearly completed sequencing 2.7 kb of the longest clone. The derived amino acid sequence is 40% identical to mouse talin over a stretch of 375 residues. On a Northern blot it hybridizes to a message of approximately 7 kb which is large enough to encode a protein of approximately 200 kd, the size of vertebrate talin. We have made a translational fusion between the putative talin cDNA and E. coli maltose binding protein, and purified the resulting fusion protein on an amylose column. Rabbit antibodies were raised against the fusion protein. When the resulting antiserum was used against Western blots of C. elegans protein extracts it recognized a polypeptide of 200 kd. Collectively, these data support the supposition that we are working on a homolog of vertebrate talin. We have not yet determined its organization in embryos or adults in situ. We anticipate the an examination of talin in wild type C. elegans, and in the mutants described above, will allow us to determine the relevance of the in vitro biochemistry of the vertebrate protein to the function of C. elegans talin in vivo.
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[
Worm Breeder's Gazette,
1982]
I have used the following procedure to stain C. elegans embryos histochemically for AChE activity. 1. On a microscope slide subbed with a 1% BSA solution, place a few young adults into a 20 l drop of 2% glutaraldehyde solution in 100 mM maleate buffer, pH 6.0. The animals are cut with a scalpel blade at the vulva to release the embryos. 2. Very gently, a 16 X 16 mm coverslip is placed on top of the buffer drop. The slide is then immersed into a liquid nitrogen container for about 15 seconds, and the coverslip is quickly pried off with a scalpel blade. 3. Another 16 X 16 mm coverslip with its two opposite edges coated with grease is placed over the embryos. The slide is incubated in a humid chamber at O C for two hours. 4. About 10 l of the staining solution ( containing 10 mg acetylthiocholinechloride, 8.8 ml 100 mM maleate buffer, pH 6.0. 1ml 50 mM sodium citrate, 100 l 300 mM copper sulfate, and 100 l potassium ferrocyanide, added in this order) is applied at the open edge of the coverslip, and is absorbed at the other open edge with an absorbant paper ( see fig.). This step is repeated 3-4 times to ensure that all embryos have been immersed in the staining solution. The slide is then incubated in a humid chamber at room temperature for about 6 hours before light microscopy. A reddish-brown stain appears in the regions showing AChE activity. [See Figure 1] The above procedure can also be used for larvae and the adult animals. Significantly, this method reveals the same areas of AChE activity in C. elegans as in the method reported by Culotti et al. ( Genetics: 97, 281-305,1981), in which the animals are treated with 95% acetone for 3 minutes. The prominent areas showing the stain for AChE activity are, the nerve ring, lateral ganglion, ventral nerve cord, dorsal nerve cord, pre-anal ganglion, and the pharryngeo-intestinal valve cells.
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[
Worm Breeder's Gazette,
1986]
We have identified a protein in C. bines properties of the flagellar ATPase, dynein with those of the microtubule-dependent translocator, kinesin. The microtubule stabilizing drug, taxol, has been used to drive microtubule assembly in extracts of either gravid adult worms or of embryos, and the resulting polymers have been isolated by differential centrifugation. One of the microtubule-associated proteins (Mr. approx. 400K) can be extracted from the polymer by 10 mM ATP and 100 mM NaCl. This polypeptide and a Mg-ATPase activity cosediment on sucrose density gradients at approximately 20 S. The specific activity of the peak fraction is 60 - 120 nanomoles ATP hydrolyzed/milligram protein/min. ( conditions have not yet been optimized). The activity is more than 50Z inhibited by either 10 uM vanadate, 1 mM N-ethyl maleimide or by 5 mM AMP-PNP. The ATPase is enhanced 50Z by 0.2Z Triton X-100. These properties are dynein-like. When the 20 S protein is mixed with either microtubules or flagellar axonemes on slide, it promotes a nucleotide triphosphate-dependent microtubule translocation. Axonemes glide with their 'plus' ends trailing. These properties are kinesin-like. However, microtubules move at about 0.8 - 1.0 um/sec., about twice as fast as with kinesin under conditions of saturating ATP. Furthermore, the motion is ATP-specific and is blocked by either 10 uM vanadate, 1mM N-ethyl maleimide, or by 0.5 mM ATP-gamma-S. Motility is slowed but not blocked by [AMP-PNP] = [ATP]. These characteristics and the presence of an ATPase activity differ from the properties described for kinesin. The roughly parallel inhibition of the ATPase and the motility by several reagents suggests that the enzyme activity and the motility are caused by the same protein or protein complex. We therefore propose that the 400 K protein from C. rotubule translocator which possesses properties of both dynein and kinesin.
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[
Worm Breeder's Gazette,
1997]
N-ethyl-N-nitrosourea, ENU, is a likely mutagen for genetic studies of C. elegans. We are working to determine optimal buffer and storage conditions in which ENU toxicity remains low with mutagenicity similar to that of EMS. Wild type N2 C. elegans were used in tests of ENU stock and storage conditions. 50 mM ENU stock solutions were prepared in one of four solutions: M9 buffer, 10 mM acetic acid, 10 mM DMSO, and 10 mM Tris pH 6.5. All ENU solutions were diluted to 0.5 or 1.0 mM in M9 buffer and used immediately after dilution. Mutagenesis was for four hours at room temperature. The parent generation of worms did not show any noticeable difference in either lethality or complete sterility, regardless of ENU buffer conditions used. We therefore used Po brood size following mutagenesis as a measure of ENU toxicity. The data show that immediate use of 1.0 mM ENU diluted from a fresh ENU/acetic acid stock provides the highest average brood size at 143 F1 per mutagenized hermaphrodite. This is approximately a two-fold reduction in normal brood size. Results obtained using ENU stock both initially prepared and diluted in M9 are similar, 124 F1/hermaphrodite. Use of ENU stocks prepared in DMSO or Tris appears to be much more toxic as average brood sizes are twenty-fold lower than normal. In an effort to reduce worker exposure to powdered ENU, we undertook experiments to determine whether ENU solutions could be stored without increasing toxicity. Experiments with M9, acetic acid or DMSO as initial solvents indicate that, over time, ENU breaks down to a molecule more toxic to worms. Average brood size drops, sometimes severely, when ENU stock solutions are kept at -20C for as little as two days. ENU stock solutions prepared in Tris were not included in this portion of the study as brood sizes were already remarkably low after immediate use. M9 buffer and DMSO solutions had the largest decreases in brood size. The average brood size resulting from a 1.0 mM ENU solution diluted from an M9 stock dropped from 124, when used immediately, to 39, when mutagenesis was performed after storage for only 2 days at -20C. Use of acetic acid as the initial solvent provided the most suitable environment for storing ENU. After immediate use, the average brood size was 143; after two days of storage at -20C the average brood size had dropped only to 119, a 17% reduction. After eight days of storage, average brood size was further reduced to 34 F1/hermaphrodite. Thus, acetic acid is the optimal solvent for storage of ENU stock solutions for short periods of time only. We previously found that ENU prepared in M9 buffer induces mutations at the same frequency as does EMS. We are currently testing the reversion frequency induced by ENU made from 10 mM acetic acid stocks. In addition, we plan to try reducing the amount of time worms are exposed to ENU.