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note
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2 embryos were screened by epifluorescence microscopy for axon scaffold defects and by Nomarski microscopy for general morphological defects. The isolated mutant strains were back-crossed against wild-type (N2) five times to remove other possible mutations. The ur41 mutation was mapped relative to lon-3(e2175) and unc-76(e911). From the Lon non-Unc recombinants, 21 of 97 segregated ur41. Subsequently, ur41 was mapped relative to lin-2S(n545) and unc-76(e911). One of six Lin non-Unc recombinants segregated ur41, which placed nid-1 to right side of lin-25. By complementation test with chromosomal deficiencies, arDf1 deleted nid-1(ur41) but not odr-3. These results ordered ur41 between lin-25 and odr-3 of linkage group V, spanned by 28 genomic cosmids (C. elegans Genome Project). Pools of three to four cosmids were injected at 5 to 10 μg/ml per cosmid along with the dominant transformation marker, pRF4, which contains rol-6(su1006) (29) at 100 μg/ml into ur41 animals carrying a dorsal sublateral nerve GFP marker transgene, kyls123(zcZ1.2::GFP) (30). The nerves were examined for the rescue of the nid-1(ur41) phenotype. Germ line transformation with one cosmid F54F3 (accession number Z79696) completely rescued the ur41 phenotypes. An 11-kb PCR product that included the entire predicted coding sequence for the nidogen gene and 2.5 kb of 5′ flanking sequence also rescued the phenotypes.
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Transgenic strains were generated by standard methods (36). pIM#194, an expression construct for nid-1, was constructed by cloning the 2.5-kb 5′ flanking region of nid-1 into pPD 95.77 vector (from A. Fire). This GFP construct was coinjected at 10 μg/ml with pRF4 at 100 μg/ml To establish a stable line, IM329 urls151 [pIM#194, pRF4]. transgenes were integrated by γray irradiation. For the ectopic expression construct of nid-7. constructs pIM#195, pIM#196, and pIM#197, were made by using the 7-kb genomic nid-1 coding region, which was amplified by high-fidelity PCR, ligated to Nhe I-Bgl II-digested vectors, pPD96.41, pPD49.83, and pPD96.52 (from A. Fire). These vectors contained 5′ flanking regulatory sequences of mec-7, hsp16-41, and myo-3, respectively (36). The unc-119 regulatory sequence was amplified by using pIM175 as template (23), and cloned into the pPD49.26 vector (from A. Fire) to construct pIM#198. These constructs were injected at 10 μg/ml, with pRF4 into nid-1(ur41); kyls123 (zc21::GFP) animals. The resulting strains are IM330 urEx153 [pIM#195]; nid-1(ur41): kyls123(zc21::GFP); IM331 urEx153 [pIM#196]; nid-1(ur41); kyls123 (zc21::GFP), IM332 urEx154 [pIM#197]; nid-1(ur41); kyls123(zc21::GFP), IM333 urEx155 [pIM#198]; nid-1(ur41); kyls123(zc21::GFP). Ectopic expression of nid-1 was checked by in situ hybridization. IM331 embryos collected 1 to 6 hours after being laid were heat-shocked at 29.5°C for 1 hour. After heat shock, embryos were placed at 20°C, grown to the L4 stage, and examined for mispositioned nerves. Of those treated, 96% (n = 54) of the animals had wild-type nerves. For IM330, IM332, and IM333 animals, 32% (n = 41), 53% (n = 72), and 97% (n = 71) have wild-type nerves. Detection of RNA in whole-mount C. elegans embryos was performed as described (37). AP-anti-Dig antibody was used for alkaline phosphatase (AP)-mediated detection. 4′,6′-Diamidino-2-phenylindole (DAPI, 1 mg/ml) was included in the staining solution to allow nuclei to be identified by epifluorescence microscopy.
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We thank C. Bargmann, J. Culotti, E. Hedgecock, H. Hutter, and D. Pilgrim for generously providing GFP marker strains, P. Yurchenco for providing the nidogen antibodies, D. Miller for providing the UNC-54 antibody, the Caenorhabditis Genetics Center for strains, A. Coulson for cosmids, M. Driscoll, G. Patterson, J. Schwarzbauer, and P. Yurchenco for comments on the manuscript, Z. Altun-Gultekin, C.-C. Huang, G. Kao, Y.-S. Lim, P. Yurchenco, and Q. Wang for helpful discussions, R. Patel for assistance with electron microscopy, and X.-C. Ren for superb technical assistance.
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