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doi: 10.1242/10.1242/dev.00341

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The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons

Okiko Uchida^1,*, Hiroyuki Nakano^1,*, Makoto Koga^1,* and Yasumi Ohshima^1,

¹ Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan

View larger version (16K): [in a new window]   Fig. 1. Mapping and cloning of the che-1 gene. (A) Genetic map position of che-1 on the linkage group I. (B) Physical map of the che-1 region. Indicated cosmids were tested for rescue of the defect in chemotaxis to NaCl of che-1 mutants. Rescue results are shown as + (positive) or - (negative), and the number of transformed lines that rescued the che-1 mutant phenotype as a fraction of the total number of independent lines tested are shown in parentheses. Each line was tested for chemotaxis at least with 20 individuals. When more than half of the animals show normal chemotaxis, the chemotaxis defect was regarded as being rescued. (C) Location of the che-1 gene. The top line and arrows show C55B7 near the left end and predicted transcripts based on the genomic sequence. PCR products and restriction fragments of the products were tested for rescue of the che-1(p696) mutant phenotype and the results are shown as in B. *The number of F1 transient transformants that rescued the che-1 mutant phenotype as a fraction of the total number of transient transformants tested.

View larger version (48K): [in a new window]   Fig. 2. Amino acid sequence of CHE-1. (A) Nucleotide sequence of the che-1 cDNA and amino acid sequence of the predicted product (CHE-1). Splice junctions were determined by comparison with the genomic sequences and are marked with vertical lines. The putative termination codon is marked with double underlines and an asterisk. The zinc-finger region is underlined. The invariant cysteine and histidine residues of C2H2-type zinc-finger motifs are boxed. Mutation sites are indicated by diamonds with resultant nucleotide and amino acid substitutions. (B) Genomic organization of the che-1 gene. Sequences encoding the zinc fingers are shaded. Seven che-1 mutations are indicated along with their respective nucleotide and predicted amino acid alterations. (C) Alignment of the zinc fingers of CHE-1 with the zinc fingers of the Drosophila protein GLASS (Moses et al., 1989). Identical residues are shaded gray. Conserved cysteine and histidine residues are highlighted. Mutation sites in CHE-1 are indicated by a black circle (nonsense) or white circles (mis-sense).

View larger version (77K): [in a new window]   Fig. 3. Expression pattern of che-1 and rescue of chemotaxis defect by che-1 constructs. (A) A che-1 genomic clone and gfp fusion constructs. Transformed lines with extrachromosomal arrays of each of these constructs in the che-1(p679) mutant background were examined for rescue of chemotaxis as in Fig. 1. GFP expression pattern was examined in wild-type background. (B,C) Overlap of DIC and fluorescent images showing expression of pche-1::gfpBglII (B) or pche-1::gfpC (C) in wild-type L2 stage animals. Scale bars: 10 µm. (D-F) Tracks of animals showing chemotaxis behaviors on an agar plate with a concentration gradient of NaCl: (D) wild type, (E) che-1(p679), (F) che-1(p679); Ex[pche-1-HindIII (che-1(+)), plin44p-gfp]. Although the che-1 mutant failed to respond to the concentration gradient, the wild-type and the rescued animals migrated to the concentration peak at the center and remained there.

View larger version (81K): [in a new window]   Fig. 4. che-1 mutation affects expression of marker genes in ASE. Fluorescent images showing expression of F55E10.7::gfp (A,B), R13H7.2::gfp (C,D), gcy-5::gfp (E,F), gcy-6::gfp (G,H), gcy-7::gfp (I,J), tax-2::gfp (K,L), ceh-23::gfp (M,N), lim-6::gfp (O,P), F25B3.3::gfp (Q,R) and DiI staining (S,T). A,C,E,G,I,K,M,O,Q,S show expression in wild-type background; B,D,F,H,J,L,N,P,R,T expression in che-1(p679) background. Arrowheads indicate ASE neurons that have no gfp fluorescence. Animals were at L1 or L2 larval stages. Scale bars: 5 µm.

View larger version (69K): [in a new window]   Fig. 5. Effect of ectopic expression of che-1 cDNA on the expression of ASE marker genes. Fluorescent images showing expression of GFP in transformant lines carrying an extrachromosomal array gpa-10 promoter-only construct (gcy-5::gfp, gcy-6::gfp and gcy-7::gfp) (A); gpa-10p::che-1 cDNA (gcy-5::gfp, gcy-6::gfp and gcy-7::gfp) (B); gpa-14 promoter-only construct (gcy-5::gfp, gcy-6::gfp and gcy-7::gfp] (C); or gpa-14p::che-1 cDNA (gcy-5::gfp, gcy-6::gfp and gcy-7::gfp) (D) in the wild-type background. Animals were at L1 or L2 larval stages Scale bars: 5 µm.

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