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[
Worm Breeder's Gazette,
1975]
In order to study the process of fertilization in C. elegans, we have been characterizing ts sterile mutations similar to the spermatogenesis defective mutants described by Hirsh and Vanderslice. These are isolated as ts sterile hermaphrodites that lay unfertilized oocytes which can be fertilized by wild-type sperm. One such mutant,
ts17, was found to have a second ts phenotype. When grown from eggs at 25 C, all 'males' homozygous for
ts17 have a normal looking male bursa but have hermaphrodite-like gonads. They are sterile. We call these mutant animals intersexes. The gonads in an intersex resemble hermaphrodite gonads to varying degrees. Commonly, intersex animals have two gonads with several mature oocytes as well as a vulva. From temperature shift experiments, the critical temperature for both phenotypes, hermaphrodite sterility, and intersex is during the first or second larval stage. The mutation is not sex linked. A second ts mutation with the same dual phenotype appears to be allelic to
ts17 so mutations in this gene may be common. Other hermaphrodite sterile mutants do not have the second phenotype.
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[
Worm Breeder's Gazette,
1975]
Following methods similar to those described by Hirsh and Vanderslice (1976), we have isolated more than 100 ethyl methane sulfonate induced temperature sensitive (TS) mutants. All these mutants propagate at 20 C (permissive temperature) and do not propagate at 25 C (nonpermissive temperature). These mutants fall into four broad classes: 1) mutants blocked in embryogenesis (over 30 mutants); 2) mutants blocked in fertilization (nearly 30 mutants); 3) mutants defective in gonadogenesis (over 30 mutants); 4) mutants accumulating in one of the postembryonic larval stages (over a dozen mutants). From this pool of mutants, we have chosen to analyze those mutants that are blocked in fertilization and those that are blocked in embryogenesis. Of the 30 or more embryonic mutants that we have examined, mutants are found which produce embryos blocked in the 1-2, 12-16, 20-30, 40- 60, and 100-150 cell stages. Twelve mutants which we have tested so far lay embryos that appear to be uniformly blocked in one particular cell stage at restrictive temperature. Their phenotype is recessive to wild type. From temperature shift experiments, we have found that the temperature sensitive period in the mutants varied from the first larval stage to the adult animal. We have also asked whether the embryonic defects of these mutants show a maternal effect. Our criteria for maternal effect is the same as that described by Vanderslice and Hirsh (1976). 1) When a homozygous TS/TS hermaphrodite is mated at nonpermissive temperature with wild-type males (+/+), no viable progeny should be produced. 2) When a heterozygous hermaphrodite (+/TS) is grown at the nonpermissive temperature for the TS mutation, one fourth of the progeny produced should be homozygous for the TS mutation. A dozen embryonic mutants blocked in the 1-2, 12-16, 20-30, 40-60, and 100-150 cell stages have been characterized. We have found they all exhibit a maternal effect. Two mutants that die after the 150 cell stage may not show a maternal effect. Since the spectrum of the mutants that we tested probably represents the general developmental stages of the worm and since all mutants so far tested exhibited maternal effect, one can conclude that maternal genome plays a major role in the embryonic development of C. elegans at least through the 150 cell stage.
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[
Worm Breeder's Gazette,
1977]
The entire embryonic cellular lineage which give rise to the intestine of C. elegans, a free living soil nematode, has been determined by light microscopic observation from the fertilized egg to the newly hatched animal. The cells which give rise to the intestine undergo both symmetrical and assymetrical cellular divisions. Besides cellular lineage analysis the cells which produce the intestine were studied for biochemical differentiation. It appears that the gut cells biochemically differentiated well before the intestinal tract is formed. The completely developed embryonic intestinal tract is composed of 20 cells and the time course for this development is about nine hours at 20-22 C. The analysis of wild-type development will provide us with the background in the study of gut embryonic mutants.
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[
Worm Breeder's Gazette,
1979]
Strains are grown on two to three 100 mm Petri plates until bacteria are nearly exhausted and a large population of L1 and L2 animals are present. The animals are washed from the plates in a 50:50 mixture of M9 buffer (or M9 medium) and B medium to a volume usually of 2.5 to 5 ml. Equal aliquots are placed into freezing vials. (Two ml plastic screw top vials of 38x12.5 mm are found to be convenient.) The vials are placed into a box made of good insulating material, e.g., polystyrene foam, bored with holes that snugly fit the vials, so that freezing may proceed at no more than 1 C per minute. [See Figure 1] The box is then put into a freezer of -80 C or lower for several hours before vials are finally stored in a liquid N2 tank. If a gas phase liquid N2 tank is available, the box may be placed there with or without prior freezing in a freezer. This method allows freezing of hundreds of vials at a time. B medium: per liter, 5.7 g NaCl; 50 ml KH2PO4 (1M, pH6.0); 300 ml Glycerol (100%); 650 ml distilled water.
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[
Worm Breeder's Gazette,
1979]
Mutagenesis is hazardous not only to animals to be mutagenized, but to ourselves as well. We, being carcinogen-/mutagen-phobics, have developed a procedure that would alleviate some hazard as well as phobia, by mutagenizing a large stock of N2 or other strains and freezing them away for future use. Strains to be mutagenized are grown on Petri plates until bacteria are nearly exhausted, that is, just before the end of the exponential growth phase. The worms are washed from the plates and treated with ethyl methanesulfonate (EMS) as described by Brenner (1974). The mutagenized worms free of mutagen are frozen in liquid N2. They are thawed out as needed. A good batch of mutagenized stocks can be permanently stored. Most survivors are younger than L3. Forward mutation rate, as measured by dumpy and uncoordinated worms, indicates that such survivors remain a good source of mutants. This procedure should also work with other mutagens.
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[
Teratog Carcinog Mutagen,
1982]
Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. [3H]phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound.
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J Biol Chem,
1999]
Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.
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[
International C. elegans Meeting,
1999]
In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.
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[
J Biol Chem,
1999]
We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.
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[
International C. elegans Meeting,
1977]