Organization of the Ptp4E gene and protein, and evolutionary analysis. (a) The EP425 element that was used to generate the ptp4E1 allele was inserted 1,157 nucleotides upstream of the transcription start site. The portions of the gene deleted in ptp4E1 and Df(1)ovo4 are shown with a solid line when confirmed by PCR, and with a dotted line to show that the endpoint lies within the dotted region. Df(1)ovo4 is the smallest deficiency that removes Ptp4E. (b) Domain organization of Ptp4E protein, and the segment that was included in the 4E-AP construct. Ptp4E consists of a hydrophobic region (HD) and 11 fibronectin type III repeats (FN-III) in the extracellular (XC) domain, and a single putative catalytic phosphatase domain in the intracellular region. The 4E-AP construct that was used to express protein consists of a secretion signal peptide sequence (pink), 6× His residues (blue) followed by Ptp4E XC domain and human placental alkaline phosphatase sequence. (c) Comparison of Ptp4E ad Ptp10D protein structure arrangement and sequence comparison. Ptp4E and Ptp10D are very similar in structure and are 54% identical along the entire length of the protein. (d) Phylogenetic tree indicating that non-drosophilid species have only one copy of Ptp4E/Ptp10D, which is more similar to Ptp10D than to Ptp4E. Ptp10D is likely to be the ancestral copy. This is visualized in the tree by comparing the branch lengths connecting Ptp4E and Ptp10D to other insect species. D. melanogaster Ptp10D is closer than Ptp4E to the sequences in other insects that have only one type III RPTP gene, indicating greater sequence similarity. Also, the Ptp4E sequence evolved much faster than the Ptp10D sequence after the duplication (compare the branch lengths within the melanogaster/ananassae/virilis cluster for the two genes). A. gambiae, mosquito; A. aegypti, mosquito; A. mellifera, honey bee.