Activity of Troponin I–lacZ promoter constructs bearing various deletions, in context with sequence identity and exon structure of the locus. (a) Plot of sequence identity between C. savignyi and C. intestinalis in the promoter construct pro.1793 (cf. Fig 1a). Green shading represents exons, the blue bar represents the MSRR, and the red bars indicate highly constrained regions (90% id; >20 bp). (b) Activity of deletion constructs, with tn.pro.1793; black lines denote sequence present in the constructs. The table lists activities of the constructs as explained below. Truncation of 329 bp from the 5′-end of the tn.pro.1793 construct completely eliminates expression in the tail muscle. Deletion of internal regions, including annotated exon 1, exons 1 and 2, and exons 1–3, has no effect. The gray line (tn.ci.pro.921) denotes the construct derived from C. intestinalis. (c) Activity of minimally sufficient regulatory region (MSRR) constructs. Fusion of the 5′-most 363 bp of tn.pro.1793 to a heterologous Forkhead basal promoter has strong activity. Deletion of the Forkhead basal promoter has no effect on lacZ expression. The reversed insert has no activity. (d–f) Relative activities of constructs. We classify constructs as (d) +++, representing 50%–100% animals staining, with many of the animals staining strongly in a majority of the tail muscle cells; (e) ++, representing 25%–50% of the animals staining in the tail muscle, and the minority of these staining a majority of the tail muscle cells; and (f) +, with <25% of the animals staining, and none of the animals staining the majority of the tail muscle cells; “-” indicates a construct that never showed staining. Constructs that were mostly “-” but stained weakly on a single occasion are denoted +/-.
