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Standard germline transformation (7) was used to generate transgenic animals carrying individual or pooled cosmids spanning the interval between mes-6 and fem-3.
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The primers 5′-CTAAAGTTAATCATACTCTGG and 5′-ACATTTTTACTTTATCAGTGG were used to amplify a 1-kb fragment that was used to probe a cDNA library in λZap. A full-length 1.5-kb cDNA was sequenced on both strands with the use of overlapping primers.
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note
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A Northern blot containing mRNA derived from mixed-stage wild-type hermaphrodites was hybridized with the full-length cDNA probe by standard methods (2).
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The unc-129 minigene was constructed by subcloning a Bgl II-Sal I 5′ promoter fragment derived from the genomic clone into the Bgl Il-Sal I site of the cDNA clone.
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3543022212
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note
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The primers 5′-GCGGCGGATCCAGGAAAATTGGAAGAGCAACCAAG and 5′-GCGGCCTGCAGTTACTTTATTCAGTTGGTTCTATG were used to amplify unc-129 coding region from DNA of ev554, ev557, and ev566 mutants and wild-type animals for sequencing.
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27
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3543029453
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note
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Axons of DA and DB motorneurons were examined by fluorescence microscopy with the unc-129::gfp reporter pAC12 (5, 23) passed genetically into daf-1 and daf-4 mutants and a newly identified TGF-β receptor mutant (18).
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28
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0026766321
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A fragment of the unc-129 5′ regulatory region extending 4.8 kb upstream from the initiator methionine codon was amplified by polymerase chain reaction (PCR) from a genomic clone (pAC3) with the primers 5′-CATTTTCTTGCTTGCTCTTCC and T7. Transcriptional reporters were made by inserting the 4.8-kb regulatory region upstream of, and in frame with, the GFP coding sequence contained in vectors pPD95.79 and pPD95.70 (+NLS) to create pAC9 and pAC10, respectively. pAC9 was cut with either Xba I, Sma I, or Bst EII Xba I and religated to generate GFP reporters containing 3 kb (pAC11), 2.5 kb (pAC12), or 3 kb with an internal deletion of 1.2 kb (pAC13) of unc-129 regulatory sequence, respectively (Fig. 3). Extrachromosomal arrays carrying unc-129::gfp reporters were created by standard germline transformation (7) and chromosomally integrated with the protocol described by M. Hamelin, I. M. Scott, J. C. Way, and J. G. Culotti [EMBO J. 11, 2885 (1992)].
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3543030073
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unpublished data
-
The neuron-and muscle-specific promoter derivatives express in essentially the same set of cells as full-length promoter derivatives (see text), except for muscle and neurons (plus seam cells), respectively. Neither derivative appears to disrupt embryonic expression. Expression of unc-129(+) from the unc-129 neuron-specific promoter does not rescue motoraxon guidance defects of unc-129 (ev554) (45% DA and DB defects collectively, n = 96), but expression from the muscle-specific promoter does rescue (3% DA and DB defects collectively, n = 88). VD and DD motoraxons are also visibly rescued by expression of unc-129(+) from the muscle-specific promoter (A. Colavita, S. Krishna, H. Zheng, R. W. Padgett, J. G. Culotti, unpublished data).
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thesis, King's College, Cambridge
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3543006779
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note
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UNC-129 was HA epitope (YPYDVPDYASL) tagged immediately after residue 291 by use of a PCR-mediated protocol. The tagged gene was subcloned downstream of the myo-3 promoter found in pPD96.52. Extrachromosomal arrays were created by standard germline transformation (7), and immunostaining was performed as described (29).
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unpublished data
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This hypothesis is supported by the identification of a gene that acts upstream of unc-129 to repress its activity in ventral body wall muscle (B. Nash, A. Colavita, J. G. Culotti, unpublished data).
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3543035338
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note
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Strains carrying unc-5::gfp or unc-40::gfp transgenic arrays were passed genetically into unc-129(ev554). Extrachromosomal arrays carrying unc-6::HA were created by coinjecting pIM97 (14 μg/ml) (29) and carrier plasmid DNA (80 μg/ml) into unc-6(ev400) and unc-129(ev554);unc-6(ev400) hermaphrodites.
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note
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We thank A. Coulson for providing cosmids; A. Fire, M. Driscoll, and D. Pilgrim for reporter constructs; R. Barstead for a cDNA library; R. Steven for the Northern blot; and D. Merz, M. T. Killeen, J. Segall, A. Spence, and H. D. Lipshitz for reviewing the manuscript. The Caenorhabditis Genetics Center, supported by the NIH National Center for Research Resources, provided strains for this work. This work was supported by grants from the NIH (R.W.P.) and from the Canadian Medical Research Council (J.G.C.).
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