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Figure 2

Figure 2. From: Genetically targeted cell disruption in Caenorhabditis elegans.

Fusion of a promoter of interest to mec-4(d) verifies promoter activity profiles and creates behavioral defects. (a) β-galactosidase staining of candidate ASH, OLQ, and IL1 neurons in animals harboring a pdel-2lacZ gene fusion. (b) Nomarski differential interference contrast microscopy image of vacuolated cells (indicated by white arrows) in a transgenic L2 animal harboring pdel-2mec-4(d). (c) Assay of nose touch responsiveness in transgenic animals harboring pdel-2mec-4(d). Transgenic lines also include pRF4, which encodes dominant rol-6(su1006). Dark gray bars indicate assay in the wild type background; light gray bars indicate assay in the mec-6(u450) background. Scores are the average of 100 animals per line, 10 trials per animal, for two independently derived transgenic lines. Error bars indicate the SD.

S. Harbinder, et al. Proc Natl Acad Sci U S A. 1997 November 25;94(24):13128-13133.
Figure 3

Figure 3. From: Genetically targeted cell disruption in Caenorhabditis elegans.

mec-4(d) vectors for transcriptional and translational gene fusions. pABL1, pABL2, and pABL3 enable translational fusions in any reading frame to be constructed. They differ in the nucleotides corresponding to the BclI site in pABL1; the BclI site is present only in pABL1 (this site is blocked by dam methylase and can only be used when DNA is prepared from a dam host). Unique restriction sites that facilitate cloning are indicated. The mec-4(d) genomic sequence includes a conserved poly(A) addition site. The unc-54 3′ end cassette (24) is positioned after this site. Note that the unique Sse8387I site in the polylinker enables fragments with PstI ends to be inserted. In general, previously constructed lacZ and GFP fusion constructs made in standard C. elegans vectors (24, 28) can be readily converted to mec-4(d) fusion constructs. Swapping XbaI–ApaI or XbaI–SplI reporter + 3′ end cassettes might be the most common conversion strategy; for constructs without the nuclear localization signal, substituting an XbaI–ApaI fragment from pABL2 into the AgeI–ApaI region of the fusion construct should produce an in-frame mec-4(d) fusion equivalent to the original lacZ or GFP fusion.

S. Harbinder, et al. Proc Natl Acad Sci U S A. 1997 November 25;94(24):13128-13133.
Figure 1

Figure 1. From: Genetically targeted cell disruption in Caenorhabditis elegans.

Effects of ectopic expression of mec-4(d). Animals are oriented with anterior to the left and dorsal to the top, except for embryos. (a–f) Nomarski differential interference contrast microscopy images (Bar = 10 μm). (g–h) Bright-field images (Bar = 50 μm). Most prominent vacuoles, which typically appear as crater-like, swollen, membrane-bound units, are highlighted by arrows. Vacuoles may be evident in several different focal planes, and thus not all appear in sharp focus. In early stage degenerations, swollen nuclei can be observed within cells (for example, c, black arrow; see ref. 8). (a) Extensive vacuolation in a heat shocked L1 animal harboring phsp-16mec-4(d). (b) Heat shocked post-pretzel stage embryo harboring phsp-16mec-4(d). (c) Ventral cord neurons swelling in the posterior of an L1 stage animal harboring punc-4mec-4(d); black arrow indicates a cell with a clearly swollen nucleus inside. (d) Small vacuolated regions in the hypodermis of an L2 animal harboring pmec-5mec-4(d). Note that hypodermal vacuoles are always small and nuclear inclusions within these vacuoles have not been observed. (e) Vacuolation of a pharyngeal muscle in an L2 stage animal harboring pmyo-2mec-4(d). (f) punc-54mec-4(d)-induced swelling of body wall muscle cells near the pharynx in an L3 stage animal. Indicated are several vacuoles along the dorsal side; the ventral side also exhibited small vacuoles in this animal that are not fully in focus. In body wall muscle, individual cells often appear to have multiple small vacuoles as shown. (g) The hypercontracted phenotype of an animal bearing the punc-54mec-4(d) transgene. (h) A mec-6(u450) mutant bearing the punc-54mec-4(d) transgene is not dramatically hypercontracted.

S. Harbinder, et al. Proc Natl Acad Sci U S A. 1997 November 25;94(24):13128-13133.

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