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[
Worm Breeder's Gazette,
1996]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to bea most effective treatment of manic-depressive illness. Although several models have been proposed to explain Li action, the molecular mechansm remains unclear. The most widely accepted model is the inositol depletion hypothesis, in which Li is thought to affect inositol phosphate turnover by inhibiting inositol monophosphatase, thus resulting in the depletion of endogenous inositol. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 C and observed the laid embryos. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants, which propagated on Li-containing medium. So far, I obtained one mutant. The mutation was tentatively assigned to chromosome V. Preliminary genetic analysis showed that the mutation showed maternal effect. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. I am now trying to isolate other mutants and also to identify early defects of embryogenesis caused by lithium treatment. I would like to thank J. Miwa for encouragement and discussions.
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[
Worm Breeder's Gazette,
1992]
Characterization of the axonal guidance and outgrowth gene
unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108
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[
Worm Breeder's Gazette,
1994]
Mutations that enhance
glp-1 identify genes required for various aspects of germline development. Eleanor Maine, Li Qiao, Jim Lissemore-, Pei Shu, Anne Smardon, and Melanie Gelber. Biology Dept., Syracuse University, Syracuse, NY 13244 and Biology Dept., John Carroll University, Cleveland, OH 44118.
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[
Worm Breeder's Gazette,
1994]
Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911
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[
Worm Breeder's Gazette,
1986]
Our aim is to obtain peptides of known sequence from Ascaris in order to determine their mode of action using electrophysiological methods. Since McIntire and Horvitz (C. elegans CSH Abstracts 1985) showed that cholecystokinin-like immunoreactivity (CCK-LI) is present in certain neurons in C. elegans, we are initially investigating the role of a CCK-like peptide (CCK-LP) in the nervous system of Ascaris using anti-CCK8 antisera to detect and localize CCK-LI. Using the methods developed by Johnson (see Johnson and Stretton, Soc. Neurosci. Abstr. 9: 302; Sithigorngul, Johnson and Stretton, C. elegans CSH Abstracts 1985) we find that in Ascaris, CCK-LI is concentrated in 2 cells (AVF cells) in the ventral nerve cord, in 3 cells in the ventral ganglion, in 4 processes in the ventral cord, and in 2 processes in the lateral line. Thus the CCK-LP is concentrated in a small minority of the 180 neurons present in the anterior region of adult Ascaris.We have developed a procedure for extracting the CCK- LP from C. elegans and fractionating the extract on a C18 cartridge. High voltage paper electrophoresis shows that added radioiodinated CCK8 is chemically intact after this extraction and after fractionation. The CCK-LP was separated from more hydrophilic components with a 20-40% gradient of acetonitrile on reversed phase HPLC. RIA's detected CCK-LI associated with a peak of optical density. Addition of authentic CCK8 (non-sulfated) to the sample showed that the RIA-positive peak was close to, but distinctly separate from, CCK8. Assuming that the specific immunoreactivity of nematode CCK-LP and mammalian CCK8 is the same, we can obtain 100 pmoles from 60g of C. elegans. From this crude estimate, it seems that the levels of recoverable peptide are sufficient for amino acid sequence determination which is now our top priority.
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[
Worm Breeder's Gazette,
1995]
It is well known that low temperatures can prolong longevity of different animals. In this study the experimental worms were mantained in liquid medium with E. coli in +21 C during the day (8-20 hrs) and in +4 C during the night, in darkness. One control group was mantained in +21 C and other control group was mantained in +4 C constantly. The obtained results are presented in the following table. ......................................................................... Control group Experimental Control group (+21C) group (+4C) Mean +/- S.D. Mean +/- S.D. Mean +/- S.D. .......................................................................... Mean longevity (days) 19,86 +/- 1,63 22,96 +/- 1,57 38,30 +/- 2,72 (n = 22) (n = 24) (n = 22) Maximal longevity (days) 34 35 50 Minimal longevity (days) 6 10 5 Mean fecundity 76,91 +/- 4,54 54,33 +/- 3,32 4,45 +/- 2,07 (n = 22) (n = 24) (n = 22) Maximal fecundity 118 95 46 Minimal fecundity 33 25 0 .................................................................... It can be concluded that such intermittent temperature is not able to prolong the life-span of C. elegans significantly, in comparison with constant cold, as well as fecundity. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (wild line) and E. coli OP50.
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[
Worm Breeder's Gazette,
1995]
Collagen is the major structural component of nematode cuticles. In Caenorhabditis elegans, the N-terminal regions of cuticle collagen proteins contain the highly conserved motif, R-X-X-R, which has been predicted to contain a potential subtilisin-like proteolytic processing site required for cleavage of the collagen proteins and normal cuticle collagen function (Kramer, 1994, FASEB 8:329-336). Cuticles of adult females of the plant-parasitic nematode Meloidogyne incognita contain a 76 kDa collagen which comprises over 50% of the B- mercaptoethanol-soluble cuticular proteins (Reddigari et al., 1986, J. Nematol. 18:294-302). We determined the N-terminal amino acid sequence of this major collagen and found it to be identical to the predicted amino acid sequence, starting at amino acid number 66, of the M. incognita Lemmi 5 cDNA clone (Van der Eycken et al., 1994, Gene 151:237-242) (Fig.1). A putative subtilisin-like protease recognition site was found immediately upstream of the region of amino acid homology between LEMMI 5 and the N-terminal sequence of the 76 kDa collagen (Fig.1). Our data support previous speculation about the existence of this novel method of collagen maturation and provide further evidence that this mechanism has been conserved during nematode evolution. In addition to protein processing, the expression of the Lemmi 5 gene was transcriptionally regulated: Lemmi 5-specific transcripts were present in adult females but not in eggs or second-stage juveniles. Also, Lemmi 5 analogs were present only in four Meloidogyne species, but not in C. elegans, Heterodera glycines, or tomato. .. PREDICTED LEMMI5 AMINO ACID SEQUENCE .. 1M A T L V V M P Q L Y S Q I N D L N L R V R D G V Q A .. F R V N T D S A W N D L M E L Q V A V T P Q S K P R S * N P F Q S L YR Q K RS L P D Y C I C Q P L E I N79 S L P D Y C I C Q P L E I N ............................................................ CONFIRMED N-TERMINAL AMINO ACID SEQUENCE .............................................................. Figure 1. The partial predicted amino acid sequence (residues 1-79) of the Lemmi 5 collagen gene is shown in roman letters. The putative subtilisin-like protease recognition sequence, R-Q-K-R, is boxed and the first amino acid after the proteolytic cleavage site is indicated by an *. The empirically determined N-terminal sequence of the 76 kDa from adult female M. incognita is italicized.
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[
Worm Breeder's Gazette,
1979]
To compare enzymatic activities of Bergerac and Bristol strains of C. elegans, we used the microsystem ' Api Zym ' focused by D. Monget ( 1978) and produced by the french firm API SYSTEM. Some of these tests, not yet commercialized, were a gift of API SYSTEM. The technique consists in microdishes which are impregnated with substrate and buffer ( PH is given in the table ). We introduced 0.1 ml of biological suspension ( a known number of adult worms sonicated in buffer and resuspended in distilled water after homogenization ) in the microdishes which were incubated at 37 C for 4 hours. Bacteria were grossly eliminated by washing; Afterwards their concentration was too low to give response with Api Zym. The reactions were revealed colorimetrically by 4 /oo ' Fast blue BB '. The intensity of the reaction is proportional to enzymatic activity and is noted from - to +++++ . A reaction noted +/- refers to a low but detectable response. To give reliable results, all the tests were read by the same experimenter and enzymatic activities in Bergerac and Bristol strains were performed at the same time. To date, the tests were carried out on 64 enzymatic activities. Most of the enzymes tested were hydrolases ( phosphatases,esterases, aminopeptidases, glycosidases, arylsulfatases ) and some were dehydrogenases. Some of the enzymatic activities tested seem to take a prominent part in postembryonic development. It's the case of Leucine arylamidase, which plays an important part in molting ( Rogers 1965, 1977 ), and could be also the case of glycyl-L-proline arylamidase which is implicated in the metabolism of collagen. The results are summarized in the following table. The two strains show similar enzymatic spectra, but differences can be noted, which are localized essentially on : - Trypsin ( n 8 ) - gamma L-glutamate transpeptidase ( n 38 ) - Glycyl-glycine arylamidase ( n 42 ) - L-threonine arylamidase ( n 54 ) - N-CBZ-glycyl-glycyl-L-arginine arylamidase ( n 56 ) Other differences, in a less degree, but yet detectable, are observed on: - Alkaline Phosphatase ( n 1 ) - L-histidine arylamidase ( n 33 ) - L-leucyl-glycine arylamidase ( n 45 ) - L-ornithine arylamidase ( n 51 ) - L-serine arylamidase ( n 53 ) All the differences observed in this table have been verified in two repetitions, which have similar results. But it could be argued that these differences could be due to nutritional adaptation, because these two strains, xenically cultured on the A1 medium ( for thirty years in case of Bergerac, and one year in the case of Bristol ) are associated with a different bacterial complex. [See Figure 1]
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Worm Breeder's Gazette,
1988]
In the vol.10, number 2 edition of The Wormbreeder's Gazette, we reported that paramyosin is phosphorylated in vitro under low salt conditions by an endogenous kinase in a reaction that is Ca++ independent. Thin layer chromatography indicated that paramyosin contained phosphoserine. Phosphate assays suggested that in vivo paramyosin had 1.8 moles of phosphate per mole of paramyosin. To determine the sites of phosphorylation, in vitro phosphorylated paramyosin was digested by NTCB. A 15,000 Da fragment was phosphorylated which corresponded to the N-terminal fragment according to Hiro Kagawa's sequence data. To obtain a more precise localization, in vitro labelled paramyosin was digested by endoproteinase-Lys-C. 2 labelled HPLC fractions were obtained and subjected to protein sequencing. One however yielded no sequence, and since the N-terminus of paramyosin is known to be blocked, probably by acetylation, we guessed that this peptide included the blocked N-terminus. Amino acid composition of the peptide was determined. The second peptide was sequenced with no difficulty. This sequence and the amino acid composition data of the blocked peptide were compared with H. Kagawa's DNA sequence of the paramyosin gene,
unc-15. They appear to arise from, and together define the N-terminus of paramyosin. [See Figure 1] With three prolines, two glycines, and eight serines(*), the N- terminal region that we have determined is decidedly not alpha-helical coiled-coil in structure and is substantially different then the remainder of C. elegans paramyosin (Kagawa's sequence) and myosin rod sequences. It would be interesting to determine what role the phosphorylation of the non-helical N-terminus of paramyosin plays in the interaction between paramyosin and other proteins in C. elegans' thick filament formation and function.