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[
Worm Breeder's Gazette,
1992]
Background. The extracellular matrix (ECM) plays an important role in maintaining the structural integrity and cellular functions of a multicellular organism. ECM components, including collagens, fibronectin, laminin, vitronectin, tenascin, entactin, and proteoglycans, have been identified and characterized in mammalian tissues. However, the structure and organization of these components in the intact matrix remain unclear. The inability to isolate mutants that are defective in a single ECM component in higher organisms impedes this type of analysis in vivo. With the advantage of the well-characterized genetics in C. elegans, we initiated a project to study the organization and structure of ECM using C. elegans as a model system. Our approach was to generate and characterize a panel of monoclonal antibodies against C. elegans ECM to use as markers for structural studies including immunofluorescent microscopy. In the future, we plan to use these monoclonal antibodies in affinity chromatography for the purification of ECM components and to use as probes for isolating cDNA. Experimental Approach. The ECM components were extracted from a mixed population of adult and juvenile C. elegans using the procedure shown in Figure 1. [See Figure 1] BALB/c mice were immunized intraperitoneally with 100 g of ECM extract homogenized in complete Freund's adjuvant followed by three bi-weekly injections of 50 g of ECM extract in incomplete Freund's adjuvant. Spleen cells from immunized mice were fused with myeloma cells and plated at a concentration of 2.5x10 +E5cells/well. Results. Of the 1,200 wells screened.approximately 32% of the hybridoma supernatants tested positive in ELISA against C. elegans ECM extracts. Supernatants from positive wells were tested by immunoblotting against C. elegans ECM extracts and by immunofluorescent microscopy on whole or fragmented C. elegans Protein species ranging from 20,000-180,000 daltons were detected by the hybridoma supernatants in immunoblotting, and supernatant recognition ranged from complex patterns of multiple bands to a single band. Immunofluorescent studies also revealed diverse staining patterns, which included staining between muscle and hypodermis, around muscle bundles, in layers surrounding intestines and gonads, and around the cuticle. Seven of the hybridomas were purified by limiting dilution cloning and injected into mice for ascites tumor production. We would like to make these monoclonal antibodies available to interested investigators in the near future. (This project was part of a course, Biotechnology Laboratory: Molecular Recognition (BSC 352), which was composed of 70% graduate students and 30% undergraduate students, and was supported by the Department of Biological Sciences and the College of Arts and Sciences at Illinois State University. We also would like to thank Robert Barstead for providing worm fragments and helpful discussion.)
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[
Worm Breeder's Gazette,
1996]
Protein phosphatases (PP) are significant regulatory enzymes of all eukaryotes. They must be present in C.elegans as witnessed by DNA-deduced PP-like amino acid sequences unraveledin the framework of the genom project. About half of them belong to Ser/Thr PP, while the rest is Tyr PP. For the assay of the main Ser/Thr PP classes, namely PP1, PP2A, PP2B and PP2C Cohen and coworkers suggested a straightforward and relatively simple procedure (1) that was adopted for the analysis of the worm. Rabbit muscle phosphorylase a phosphorylated by phospho- rylase kinase, rabbit muscle inhibitor-1 and casein from bovine milk phosphorylated by bovine heart cAMP-dependent protein kinase were used as substrates. C.elegans Var. Bristol (N2) wild type strain were cultured, harvested, rinsed with the extraction buffer, frozen in liquid nitrogen and homogenized in four volumes of the ice cold extraction buffer (50 mM Tris-HCl pH=7.4; 0.1 mM EDTA; 10 mM DTT; 0.5% Triton X-100; 2mM PMSF; 5mM benzamidine and 1 mM o-phenantroline).The homogenates were cleared by cent- rifugation at 14000 g, 4 oC for 10 min and than were kept frozen in liquid nitrogen till the assays. Rabbit skeletal muscle inhibitor-2 (unphosphorylated) was used as a specific inhibitor ofPP1 with 32P-phosphorylase a substrate in the presence ofEDTA,a metal ion chelator(Fig.1).The titration curve reveals that about 75%of the total activity was supplied by PP1 and 5 nM of inhibitor-2 caused about 50% inhibition. Okadaic acid, a potent marine toxin and tumor promoter, was used to separate PP1 and PP2A activities with 32P-phosphorylase a substrate in the pre- sence of EDTA (Fig.2). The titration curve with okadaic acid is bi- phasic, in the first step about 30% of the total activity is inhibited with an IC50 of ~0.04 nM and the rest of activity was inhibited in a second step with an IC50 of ~70 nM. Complete inhibition was achieved by 1 micromol/liter okadaic acid concentration. It is known that PP2A is more sensitive to okadaic acid than PP1, thus the first step of the titration can be explained by the presence of PP2A while the second step can be attributed to PP1 inhibition. For the detection of PP2B we used 32P-inhibitor-1 substrate and Ca-calmodulin as an activator. Due to the interference of prote- ases the 32Pi liberated in the assays was estimated after ammonium molybdate extraction(2). About 50 nmol substrate was converted in 1 min by the extract. ~70% of the activity was inhibited by 4 mM EDTA or 1.5 mM trifluoperazine and ~30% was inhibited by 2 nM okadaic acid (not shown). Thus 70% of the total activity was due to PP2B and 30% to PP2A. PP2C was assayed with 32P-casein substrate in the presence of Mg2+-activator. ~10%of the activity was inhibited by inhibitor-2 (PP1) ~40% of the activity was inhibited by 2 nM okadaic acid (PP2A) and ~50% was stimulated by Mg2+ (PP2C). Half maximal activation was achieved at 2 mM Mg2+ concentration, maximal activation was measured at 20 mM (Fig.3.). Acknowledgements.This work was supported by the grants OTKA 6005 and 12840. (1) Cohen, P., Klump, S., Schelling, D.L. (1989) FEBS Lett. 250, 596-600. (2) Shenolikar, S., Ingebritsen, T.S. (1984) Meth. Enyzmol. 107, 103-129.
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[
Worm Breeder's Gazette,
1994]
The beginning Strongyloides is an obligate parasitic nematode. It is the parasitic nematode taxonomically closest to Caenorhabditis. Its life-cycle (see below) consists of two distinct adult generations; one female and parasitic and the other dioecious and free-living. The free-living adult generation (known as heterogonic development) can be omitted and instead larvae can develop to infectiveL3 sdirectly (homogonic development). Cytological studies have concluded that the parasitic generation reproduces by mitotic parthenogenesis, and the free-living generation by pseudogamy (maternal inheritance only)(1). Here I describe a genetic analysis of the life-cycle of S. ratti. Doing genetics with Strongyloides Clones A rat can be infected with a singleL3 ,which gives rise to a single parasitic female. Larvae produced by this worm are able to develop both directly and indirectly. We refer to such infections (and the population derived from these) as clones, but they should probably be more correctly called isofemale lines. Controlled matings Virgin free-living males and females can be grown by collecting early stage larvae and maintaining them individually until they are mature. Virgin males and females from different clones can be brought together so that mating can occur. The progeny can be cloned back into rats or can be analyzed directly. Genetics of the free-living generation Crosses have been made between individual virgin males and virgin females of different clones (the parental clones). The resulting progeny of such crosses have also been cloned (the progeny clones). Parental and progeny clones have been analyzed by minisatellite fingerprinting (Jeffreys' probe 33.15)(2). The fingerprints showed that inheritance was bi-parental, and thus that pseudogamy did not occur. The inheritance patterns are fully consistent with the occurrence of "normal" sexual reproduction(3). These results were incompatible with the earlier cytological observations. Genetics of the parasitic generation Parthenogenesis can occur by a number of cytological mechanisms. In some cases, progeny are identical to each other and to their mother (mitotic and some forms of meiotic parthenogenesis). In other cases the progeny differ from each other and from their mother (other forms of meiotic parthenogenesis). In view of the quite different conclusions drawn from the cytological and genetic data, I have also analyzed the progeny of single parasitic females with respect to the functional difference (mitotic or meiotic) in parthenogenetic reproduction. Larval progeny of individual parasitic females have been analyzed for a RFLP in a PCR fragment of C. elegans actin 4 (4). Alleles of this locus segregated in a Mendelian manner in crosses of the free-living generation. Parasitic females that were heterozygous for this marker produced progeny all of which were heterozygous. Thus, it was concluded that the parasitic female was functionaIly mitotic. The genetic and cytological studies were not in conflict for this stage. The future Strongyloides is a parasitic nematode with a life-cycle that is amenable to genetic manipulation. Admittedly it isn't quite as easy to maintain or work with as C. elegans, but it is the most accessible of the parasitic nematodes. I would be happy to supply further details of the methods used or the work itself, if anyone is interested. References 1. Bolla & Roberts, 1968, J. Parasit., 54, 849-855. 2. Jeffreys, et al., 1985, Nature, 314, 67-73. 3. Viney, et al., 1993, Proc. R. Soc. Lond. B, 254, 213-219. 4. Krause, et al., 1989, J. Mol. Biol., 208, 381-392.