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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,
2002]
The availability of polymorphic strains of P. pacificus and a genomic BAC library of Pristionchus pacificus with large insert sizes encouraged us to use a BAC end based strategy to achieve our goal of constructing genetic and physical maps of the satellite model system Pristionchus pacificus . We looked for polymorphisms in the sequenced BAC ends using the SSCP ( single stranded conformation polymorphism) technique. The BAC end sequences were obtained from our Genome Centre at the institute and primers were designed to amplify 180-250 bp amplicons within these BAC end sequences. We used an inhouse program Prime Array 3.0 to design the primers. These primers were used to amplify both California and Washington DNA's and the resulting PCR products were run on an agarose gel to check for the presence or absence of bands. Later they were run on an SSCP gel to check for mobility differences. We found a total 131 SNP's . BAC ends that showed mobility differerences were then rechecked by sequencing the Washington PCR product and compared to the already available California sequence. Sequence comparison was done on a commercially available program Sequencher3.0. We found that the percentage of insertions and deletions in Pristionchus pacificus was more than in C.elegans (data from Wicks et al ) However the single base pair substitutions were lesser than in C.elegans . This result indicates that insertion and deletion events are more common in Pristionchus pacificus than single base pair substitution events.
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[
Worm Breeder's Gazette,
1982]
We are using immunofluorescence microscopy to screen mouse hybridoma cell lines for the production of monoclonal antibodies against C. elegans antigens. In the course of this work, we observed that the FITC-conjugated rabbit anti- (mouse IgG) used as secondary antibody reacted specifically with cytoplasmic components unique to the germline precursor (P) cells of embryos. Using fluorescent antibody staining, we have followed these components throughout the life cycle. We have termed them P-granules, and the antibody that stains them in P and Z cells we have called PZA. P-granules are detectable in the uncleaved zygote as prelocalized particles at the posterior pole. After the first cleavage they are detected only in the P1 cell. In subsequent divisions they are progressively segregated to the P2, P3, and P4 cells. During these early divisions P-granules become localized prior to cleavage in the region of cytoplasm that is destined for the next P cell daughter. Between the 10-cell and 16-cell stages the number, size, and distribution of P-granules change; the numerous, small cytoplasmic particles present in very early embryos appear to coalesce into larger perinuclear granules. These characteristics of P-granules are similar to those of 'nuage' seen by electron microscopy in early C. elegans embryos. At the 100-cell stage P4 divides into Z2 and Z3, which are the only cells stained by PZA in embryos from the 100-cell stage to hatching of the first stage larva. Within the developing larval gonad, PZA stains perinuclear granules that are seen in all the descendants of Z2 and Z3, but not in the somatic gonad cells. PZA staining of perinuclear granules is also seen in the distal arm of the adult gonad, where germ cells divide mitotically and then enter meiosis. As oocytes mature, the granules disperse from the nuclei. PZA stains mature oocytes diffusely; granules are sometimes observed randomly distributed in the cytoplasm. Mature sperm obtained from males show cytoplasmic staining by PZA. There is also a high level of non-gonadal staining in late larvae and adults. The cross-reactivity of PZA seems to be limited. We have tested embryos of mouse, Drosophila melanogaster, d Panagrellus redivivus. Only the Panagrellus embryos showed specific staining of what are presumably the germline precursor cells. Immunofluorescent staining of P-granules has been observed with 3 different lots of fluorochrome-conjugated rabbit anti-mouse antibody ( F-RAM) from Miles Laboratories, as well as 2 lots of fluorochrome- conjugated goat anti-rabbit antibody (F-GAR) from Miles. However, F- RAM and F-GAR from 2 other companies do not stain P-granules. The Miles F-RAM used in this study was prepared from the pooled sera of 15 rabbits that had been immunized with mouse IgG. Some possible explanations for the presence of PZA in the F-RAM are: 1) PZA may be a rabbit autoantibody to an evolutionarily conserved or cross-reacting rabbit antigen. 2) PZA may have been elicited by a contaminant in the mouse IgG injected into the rabbits as immunogen. 3) One or more of the immunized rabbits may have had a nematode infection, which elicited production of antibodies, including PZA, that cross-react with C. elegans. The third explanation is consistent with both the limited cross-reactivity of PZA with other species and the high level of general staining of larval and adult C. elegans preparations by Miles F-RAM. We recently tested the serum from 11 wild and potentially worm-infected rabbits from a local farm; screening by indirect immunofluorescence, we found that 1 of the 11 serum samples contained PZA. We are presently using the Miles F-RAM and F-GAR to ask about the composition, mechanism of segregation, and possible role of P-granules. The wild PZA-producing rabbit may help us determine the origin of the antibody.
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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.
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[
Worm Breeder's Gazette,
1999]
Mechanotransduction plays a central role in fundamental physiologic processes such as detection of touch and sound, regulation of cell volume, and control of motility. Mechanosensitive channels have been studied extensively using electrophysiology. Little is known, however, about the molecular structure of mechanosensitive channels or about how mechanical stress is transduced into altered channel gating. We developed techniques to patch-clamp and study ion channels in C. elegans embryonic cells. In cell-attached and inside-out patches, application of gentle suction activated a mechanosensitive current. Suction caused an immediate increase in current amplitude of 6.4 2.8 fold (n = 20) at +100 mV in inside-out patches. The current rapidly inactivated when suction was discontinued and could be repeatedly reactivated by additional suction. When membrane voltage was ramped from +100 to -100 mV at 100 mV/second, the current showed moderate outward rectification (outward:inward current = 2.0 0.05 at 100 mV). Current amplitude was largely unaffected when bath Na+ was replaced with NMDG+ (n = 10). However, replacement of bath Cl- with either gluconate or glutamate reduced inward and outward currents by 46 10% and 39 7%, respectively (n = 9). Replacement of 120 mM bath Cl- with a mixture of 60 mM Cl- and 60 mM SCN- increased the inward current at -100 mV by 3.0 0.4 fold and shifted Erev by 16 2 mV (n=7). We conclude that membrane stretch activates a novel mechanosensitive anion current in inside-out patches from C. elegans embryonic cells.
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[
Worm Breeder's Gazette,
1978]
We have examined the early cleavages of our ts zyg mutants by Nomarski optics and measured the number of nuclei in the embryos as a function of time at restrictive temperature using Hoechst stain. We find that all of the strict maternal mutants (M,M) either have abnormal early cleavages or never progress beyond 200 nuclei or both. Two of the other zyg mutants display abnormal early cleavages. Interestingly these are the paternal mutant B235 (M,N,P) and the anomalous mutant B261 (M,N,?). None of the other mutants displays abnormal early cleavages or stops before 200 nuclei. The first cleavages can be schematically diagrammed as follows for N2 and for some of the mutants: [See Figure 1]