Absence of whn-related genes in mouse, zebrafish, and amphioxus. Hybridization analysis was performed under low stringency conditions (6x SSC at 65°C) with cDNA probes spanning the DNA binding domain from the indicated species. DNAs were digested with HindIII (mouse), SpeI (zebrafish), and DraI (amphioxus) to reveal single fragments for the known whn genes. Note the absence of additional hybridizing bands.

Recognition of Whn binding sites in vitro and in vivo. (A) Electrophoretic mobility shift assays using mouse, zebrafish, and amphioxus DNA binding domain peptides. A double-stranded oligonucleotide obtained via in vitro selection was used in wild-type configuration (core sequence ACGC), in modified forms (changed nucleotides in core sequence are indicated by lowercase letters), or in in vitro methylated form (m denotes 5-methylcytosine). (B) Transactivation of a luciferase reporter gene after transient transfection into BHK cells. A luciferase gene with a minimal promotor (11) was cotransfected with a mouse whn (DBD^Mm) expression plasmid or with constructs in which the mouse DNA binding domain was changed to that of zebrafish (DBD^Dr) or amphioxus (DBD^Bl) to establish a luciferase baseline activity. These values were compared with reporter constructs in which a whn response element was positioned upstream of the minimal promotor and expressed as fold transactivation. Values shown represent the average of two experiments with a variation of less than 20%. I refers to expression constructs with an N-terminal MYC tag; II refers to constructs with a C-terminal MYC tag. DBD, DNA binding domain; AD, transcriptional activation domain (8). In control experiments, the transfection of whn expression constructs in anti-sense orientation had no effect on luciferase activity.