Mutant sequence analysis of the Tv4-enhancer.

<p>(<b>A</b>) Conservation track from UCSC Browser showing conservation islands between 12 sequenced <i>Drosophila</i> species. (<b>B1-B9</b>) Graphic representation of the position of putative HD (green), Mad (red and magenta) and Med motifs (yellow) as the coloured vertical bars within the Tv4-enhancer. These overlay the depicted deletion series of the Tv4-enhancer B1-B9. We generated a series of deletion transgenes (B1-B9; dotted region deleted). <b>B1</b> is the full length wild-type Tv4-enhancer, denoted by the complete horizontal solid black line. <b>B2-B9</b> show the region present in the reporter transgene (horizontal black bar) after removal of certain sequences (dotted regions). <b>B4,5,7</b> show a deletion between two regions; the points of fusion are shown by vertical bars, between which the intervening sequence is removed. <b>B8,9</b> show the type of sequence conversion performed within the region shown. (<b>C</b>) Reporter expression driven from control or deletion mutants B1-B9, or the empty reporter vector, expressed in the bar graph as the % nEYFP fluorescence intensity relative to mean of the B1 control. The right-most panel provides the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Removal of the HD-A motif (green, B3,B4) or the Mad-D motif (magenta, B3,B5) severely reduced expression. The enhancer fragment spanning the HD-A to Mad-D motifs (B6) expressed at moderate levels, but alteration of the intervening sequence severely reduced reporter expression, whether by sequence elimination (B7), conversion to complementary sequence (B8) or non-canonical sequence conversion (B9). (<b>D</b>) Point substitution mutants of Mad, HD and Med motifs, showing (bar graph) expression levels as % nEYFP fluorescence intensity relative to mean of the control, or (bottom-most panel) the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Only substitution mutants that alter the HD-A or Mad-D motifs essentially eliminate reporter expression. n = 10–20 animals per genotype. All data represented as mean±SEM. Data compared using one-way ANOVA with Tukey HSD <i>post-hoc</i> test. * = p<0.05 compared to controls.