[
Worm Breeder's Gazette,
1996]
There is precious little in the literature regarding the regenerative ability of nematodes (Poinar, 1988). The received wisdom has been that the determinate cleavage in these pseudocoelomates precludes any possibility of epimorphic regeneration (K.A. Wright pers. comm., 1988). Although Filipjev (1921) stated that regeneration is "completely absent" in nematodes, other reports by Micoletzky and Kreis (1930) and Allgen (1959) present another picture which seemingly contradicts such a blanket generalization. Last year it was reported that a gastrotrich (undescribed species of Turbanella) of the genera Macrodasyidae, a primitive sister group of the Nematoda, restored epidermis with complete wound closure following transection; restructuring of Y organ and intestine; and caudal adhesive tubes forming anew (Manylov, 1995). This is the first report of regeneration in this group. In another recent paper from the former Soviet Union, Voronov and Panchin (1995a) report that a nematode, of the order Enoplida (E.brevis), has a process of gastrulation which contradicts the patterns of cleavage formerly ascribed to the Enoplida (Malakhov,1994). They observed that up to the sixteenth cell stage cleavage is usually (though this can vary) equal and synchronous, producing blastomeres of equal appearance; elsewhere they observe that the primordia which gives rise to all the endoderm can be derived from either the anterior or posterior at the two-cell stage (Voronov and Panchin,1995b). This variability, they offer, makes the Enoplida different from other nematodes studied. Malakhov believes that this variability "can even engender the idea that the cleavage among members of marine Enoplida is indeterminate, but this is not so." (p.166). However, the cleavage of the Enoplida may be indeterminate enough to allow for the regenerative phenomena recently witnessed in a gastrotrich. In sum, Enoplid cleavage patterns would appear to be similar to the more primitive patterns seen in the Macrodasyidae, which is consistent with the notion that equal cleavage is ancestral and determination of early blastomere fate derived (Baguna and Boyer,1990). Also, it should be remembered that in addition to the single species reported by Micoletzky and Kreis, all nine of the species which Allgen found evidence of regeneration were marine Enoplids. Allgen,C.A.(1959)Free living marine nematodes. Further Zool. Results Swed. Antarct. Exp. 1901-03 vol.5 no.2: 1-293. Baguna,J.,B.C.Boyer(1990)Descriptive and experimental embryology of the Turbellaria: Present knowledge, open questions and future trends. In Marthy, H.(ed), Experimental Embryology in Aquatic Plants and Animals. NATO ASI 195; 95-128. Filipjev,I.N.(1921)Free living nematodes in the vicinity of Sevastapol. (in Russian), Akad. Nauk SSSR. Trudy osob. zool. lab. ser 2 41: 351-614. Malakhov,V.V.(1994)Nematodes: Structure, Development, Classification and Phylogeny. Smithsonian Inst. Press. Manylov,O.G.(1995)Regeneration in Gastrotricha - I. Light microscopical observations on the regeneration in Turbanella sp. . Acta Zool. 76:1-6. Micoletzky,H.,H.A.Kreis(1930)Freilbende marine Nematoden von den sunda-Inseln. Dansk natur. Foren. Vid. medd. Bd 87: 243-339. Poinar,G.O.(1992)Immune responses and wound repair. In Diseases of Nematodes. vol 1, p.133-40, CRC Press, Boca Raton, Florida. Voronov,D.A.,Y.V.Panchin(1995a)The early-stage of the cleavage in the free-living marine nematode Enoplus brevis (Enoplida, Enoplidae) in the normal and experimental conditions. Zool. Zhurn. 74(6): 31-38. Voronov,D.A.,Y.V.Panchin(1995b)Gastrulation in the free-living marine nematode Enoplus brevis and the localization of endodermal material at the stage of 2 blastomeres in the nematodes of the order Enoplida. Zool. Zhurn. 74(10): 10-18.
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Worm Breeder's Gazette,
1997]
Mammalian Ras proteins regulate multiple effector molecules. They include Raf family (Raf-1, B-Raf, A-Raf), RalGDS family (RalGDS, RGL, RGF) and AF-6. C. elegans genetic studies contributed a lot to understanding of Ras function, especially regulation of Raf-MEK-MAPK pathway. However, studies have been limited to Raf and its downstream. Our long-term goal is to understand all pathways regulated by C. elegans Let-60. Here we report progress of our search for new Let-60 effectors and genetic study of their functions. We first screened for Let-60-binding proteins by using the yeast two-hybrid system. lACT-RB2, a random-primed mixed-stage C. elegans cDNA library was constructed in lACT that express cDNA clones fused to GAL4 activator domain. After conversion to the plasmid form (pACT-RB2), it was co-transformed into the test yeast strain CG-1945 with a plasmid pAS2-1-Let-60V12, expressing an activated Let-60 mutant as a fusion with GAL4 DNA-binding domain. After screening of approximately 106 transformants, 88 colonies were identified as both His+ and LacZ+. Plasmid clones were recovered and confirmed to confer both His+ and LacZ+ phenotypes when co-transformed again with pAS2-1-Let-60V12 but not when co-transformed with pAS2-1 vector. Inserts of confirmed clones were characterized by DNA sequencing. The sequencing data were used to identify cosmid clones containing overlapping genomic sequences by the BLASTN search (thanks to the C. elegans genome sequence project). The cDNA sequences together with the cosmid sequences were also used to identify EST clones containing overlapping sequences (thanks to Dr. Y. Kohara at NIG, Japan). When corresponding cosmid was not found, the cDNA sequences or the EST sequences were used to identify homologous proteins from the protein database by the BLASTX search. The clones comprised 6 classes. They included those encoding C. elegans Raf (Ce-Raf, 35 clones), C.elegans homologs of RalGDS (Ce-RalGDS, 29 clones), of AF-6 (Ce-AF-6, 6 clones), of Cdc25 (Ce-Cdc25, 10 clones) and of phospholipase Cb (Ce-PLCb, 2 clones), and those encoding other proteins (6 clones). Since lACT-RB2 represents 107 independent clones, this screen is not saturated yet. Predicted structures of newly found Let-60 effectors are shown in Fig. 1. We found Ce-RalGDS (F28B4.2) initially by simply performing a BLASTP search with mouse RalGDSA. Two-hybrid clones contained regions encoding the middle Cdc25 homology domain, the C-terminal Ras-interacting domain and a termination codon. pACT-RB2 plasmid library was used as a template to obtain sequence for N-terminal portion by PCR with a SL1 splice leader primer and a cDNA-specific primer. Clones for Ce-AF-6 contained the initiation codon and regions encoding the N-terminal Ras-interacting domain and the middle GLGF/DHR motif (no cosmid). Sequence for the C-terminal portion and the termination codon was found in a EST clone. Clones representing Ce-Cdc25 (T14G10.2/K04D7) encoded a Cdc25 homology domain most similar to that of Cdc25Mm/RasGRF. But regions around this domain were divergent from Cdc25Mm/RasGRF. It is possible that Ce-Cdc25 is a downstream effector of Ras, not a homolog of Cdc25Mm/RasGRF, an upstream regulator of Ras. Clones for Ce-PLCb (F31B12.1) encoded the X, Y and C2 domains found in the human counterpart. The Ras-binding domain was mapped to the C-terminal 300 amino acids by deletion analysis. F31B12.1 contains a very long N-terminal extension not found in human PLCb. But this prediction seems to be correct since PCR with a primer containing the predicted initiation codon and a downstream primer using the pACT-RB2 plasmid library gave a band with a predicted size. By computer analysis, Ponting and Benjamin recently proposed the existence of a family of Ras-associating domains (RA domain) including those of RalGDS and AF-6 [1]. Their list of proteins containing the RA consensus included Ce-RalGDS, Ce-Cdc25 and Ce-PLCb. However, Ce-AF-6 was not detected in their search, since it was not contained in any sequenced cosmids. Also, the list did not include Cdc25Mm/RasGRF, supporting the uniqueness of Ce-Cdc25. Search is in progress for mutant worms carrying Tc1-insertions within these genes. A PCR-based method was used to screen 108 plates, each started with ten MT3126 worms (thanks to the CGC). Two alleles each for Ce-RalGDS and Ce-PLCb were detected (we also found one allele for the previously reported Ach-1 gene[2]). After sib-selection, single worms were identified for all of them (thanks to Dr. Y. Andachi and the Japan C. elegans laboratory course at NIG). We are in the process of screening for worms carrying deletion of these genes by Tc1-excision.