Article Figures & Data
Figures
- Figure 1.
Identification and characterization of the eol-1(yx29) mutant. A, Schematics of aversive training with P. aeruginosa PA14 and microdroplet assay. B, Histogram of binned preference indexes of trained wild type (WT) and F2 clones in the first-round screen. Bin size = 0.025; columns are slightly nudged for a clear display. SD indicates standard deviation of wild type indexes. C, D, Preference index (C) and learning index (D) of wild type and eol-1(yx29) in aversive olfactory learning assay, two-tailed Student's t test; ***p < 0.001. E, Turning frequency of wild type and eol-1(yx29) in response to the alternating smells of E. coli OP50 and P. aeruginosa PA14. Two-way ANOVA, a significant genotype × treatment interaction (***p < 0.001) was detected for responses to OP50 odor (genotype, p < 0.01; treatment, p > 0.05), but not for responses to PA14 odor (n.s., p > 0.05 for genotype × treatment interaction; genotype, p > 0.05; treatment, p < 0.001). F, Turning rate of eol-1(yx29) to the alternating smells of NGM buffer and bacteria OP50 or PA14 (two-tailed Student's t test; ***p < 0.001). G, WT and eol-1(yx29) respond similarly to the alternating smells of OP50 and NGM buffer. Two-way ANOVA (n.s., p > 0.05 for genotype × treatment interaction; genotype, p > 0.05; treatment, p < 0.001). C–G, n ≥ 9 assays, mean ± SEM.
- Figure 2.
Wild-type T26F2.3 sequence restores normal learning to eol-1(yx29). A, Genomic structure of T26F2.3 sequence (Ensemble Genome Browser, C. elegans WS220). Filled boxes denote exons. Arrowhead denotes the S285F mutation in yx29. Cosmids tested for rescue are shown. B, Expression of T26F2.3 restores learning in yx29. Transgenic animals are compared with nontransgenic siblings, n ≥ 12 assays each. C, Time course of learning. eol-1(yx29) mutants are compared with wild-type controls at each time point, n ≥ 6 assays each. B, C, Two-tailed paired t test; ***p < 0.001, **p < 0.01, *p < 0.05; n.s., not significant, mean ± SEM. D, Slow-killing assays on P. aeruginosa PA14; n = 3 assays each genotype, n ≥ 3 replicates per assay, log-rank test with Bonferroni correction, no significant difference between eol-1(yx29) and wild type. nsy-1 and daf-2 are controls for reduced and enhanced PA14 resistance, respectively. lin-15B(n765);kyIs30 is a wild-type reporter line used for backcrossing. Error bars indicate SEM.
- Figure 3.
eol-1 is expressed in neurons and functions in URX to regulate olfactory learning. A, Expression of Peol-1::gfp in a wild-type adult hermaphrodite. Arrows denote reproductive system and arrowheads denote neurons. The transgenic marker Punc-122::gfp is expressed in coelomocytes (empty arrowheads). B–E, Expression of eol-1 in hermaphrodite reproductive system (B) and in URX, AVF, and PQR neurons (C–E). The gcy-36 promoter drives expression in URX and PQR and the unc-4 promoter drives expression in AVF and SAB. F, Aversive olfactory learning of wild type, eol-1(yx29), and transgenic eol-1(yx29) animals that express wild-type eol-1 gene with cell-specific promoters. Transgenic animals are compared with nontransgenic siblings, two-tailed paired Student's t test; **p < 0.01, n ≥ 10 assays, mean ± SEM.
- Figure 4.
Mouse Dom3z cDNA rescues the learning phenotype of eol-1(yx29). A, Sequence conservation among EOL-1 homologs. *Indicates positions with a single conserved residue; : indicates conservation between groups of strongly similar properties; · indicates conservation between groups of weakly similar properties; and color-code indicates physicochemical properties of amino acids (ClustalW2.1). B, Mouse Dom3z.b cDNA rescues aversive olfactory learning of eol-1(yx29), but the mutated isoform Dom3z.b(E234A, D236A) or eol-1(E185A, D187A) does not. Wild type and eol-1(yx29) are compared using two-tailed Student's t test; n ≥ 4 assays, *p < 0.05. Transgenic animals are compared with nontransgenic siblings with two-tailed paired Student's t test; n = 14 assays, **p < 0.01, n.s., not significant, mean ± SEM. C, Model of EOL-1 function in aversive olfactory learning.
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