Supplementary figure 2. The C. elegans TRPM channels
gtl-1 and
gtl-2 are expressed in the intestine and pharynx and excretory cell respectively.A-E:
gtl-1 is expressed in the intestine. The intestine is marked by white arrows in panels A-D and a black arrow in panel E. Panels A-D show transgenic animals carrying GFP fused to part of the
gtl-1 gene created as below. A, expression in the intestine (white arrows) of an adult. B, a closer view of the intestine (white arrow) of a young adult. C an oblique section in which GFP expression in the lining of the intestine (white arrow) is clear. D, expression in the intestines (white arrows) of two three-fold embryos. E, In situ hybridization of
gtl-1 in an L1 larva, F,G:
gtl-2 is expressed in the pharynx and excretory cell. A fusion of
gtl-2 and GFP was created ina similar way to
gtl-1::GFP above (see below). F, expression in the pharynx GFP is observed throughout the muscle cells, parts of the excretory cell are also visible(arrowhead). G, expression in the excretory cell (white arrow) of an L2 larva. The canals are labeled. The animal shown in G did not show expression in the pharynx and was used for clarity. GFP fusions for
gtl-1 were constructed as follows. The 5' end of the
gtl-1 mRNA was determined by 5' RACE (unpublished). A PCR fragment extending from 4.8 kb upstream of the ATG start codon to the start of exon 2 was subcloned upstream of GFP in the vector pHAB200 [1] so that the
gtl-1 and GFP sequences were translationally fused. Similarly a PCR fragment extending from exon 30 to 2kb downstream of the stop codon of
gtl-1 was translationally fused downstream of GFP.The final plasmid, called pCK5 was microinjected into C. elegans [2] at 20 μgμl-1together with a
rol-6 marker [3] and PvuII digested C. elegans genomic DNA (75μg μl<sup>-1</sup>). Stable transformants were selected and analyzed using a Leica SP1 Confocal Microscope. Three independent lines were analyzed. GFP fusions for
gtl-2 were created in a similar way. The upstream fragment extended from 5.4 kb upstream of the ATG to the middle of exon 3. The downstream fragment extended from exon 25to 2.4 kb downstream. The final plasmid (pCK7) was injected into C. elegans at 20μg μl<sup>-1</sup> with the
rol-6 marker. In situ hybridization was carried out using the method of Mitani et al., [4] and Ogawa et al., [5].