Fig. 7. Molecular screen with minimal sequences from myoblast enhancer I-E_D5 identifies Lmd as the DNA-binding factor. (A) Diagram of deletion constructs, each of which has an internal deletion (thin line) within the 170 bp myoblast enhancer I-E_D5. Vertical lines bracket the endpoints of the essential [C/D]* region. (B) Sequence of enhancer I-E_D5. Arrows demarcate the [C/D]* region and mutated sequences in constructs I-E_D5-mt1, I-E_D5-mt2, I-E_D5-mt3 and I-E_D5-mt4 are underlined. (C) Diagram of the 12 cDNA clones encoding partial Lmd proteins from the one-hybrid screen with [C/D]* as a specific target. Unrelated target T2 was used to check for specificity. The number of clones recovered for each type is in parentheses. Lmd derivatives used to localize the DNA binding domain are also shown. The checked box denotes the Zn-finger domain. Activation of His and lacZ expression was monitored; N.D. denotes not determined. (D) DNA binding assays with in vitro translated Lmd protein and ³²P-labeled I-E_D5 probe, in the absence or presence of 50x molar excess of cold specific competitor DNA. Free probe is marked as (U). Lane 1, probe alone; Lane 2, lysate without DNA template; Lane 3, Lmd protein lysate; Lane 4, Lmd + cold I-E_D5 DNA; Lane 5, Lmd + cold unrelated III-F₇ DNA. (E-J) Transgenic embryos were stained with an anti-ß-gal antibody. Robust levels of lacZ expression are seen in somatic myoblasts of embryo with I-E_D5 construct (E), whereas embryo with I-E_D5-DelD shows a nearly complete loss of expression (F). Construct 5x[C/D]* drives expression in myoblasts similarly to parental I-E_D5 (compare E with G). Embryo with I-E_D5-mt1 (H) or I-E_D5-mt2 (I) exhibits dramatic loss of reporter gene expression in somatic myoblasts, while embryo with I-E_D5-mt3 (data not shown) or I-E_D5-mt4 (J) shows normal levels of expression. Ectopic expression in I-E_D5-mt2 embryo is not in somatic myoblasts.